LOCUS       40924_18856        9965 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Mammalian expression vector pEXPR166, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9965)
  AUTHORS   Siciliano V.
  TITLE     Engineering modular intracellular protein sensor-actuator devices
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9965)
  AUTHORS   Siciliano V.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-APR-2018) Biological Engineering, Massachusetts 
            Institute of Technology, 500 Tech Square, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 9965)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9965)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-APR-2018) Biological Engineering, Massachusetts Institute of 
            Technology, 500 Tech Square, Cambridge, MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(222..810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(984..1841)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(1842..1946)
                     /label=AmpR promoter
     rep_origin      complement(1972..2427)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     2569..2585
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2595..2613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2628..3431
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    3510..3535
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    4207..4227
                     /label=attB4
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     promoter        4252..5424
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     protein_bind    5446..5470
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      5505..5514
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5619..5648
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6102..6797
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
     CDS             7287..7520
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     CDS             7527..7964
                     /label=GAL4 DNA binding domain
                     /note="DNA binding domain of the GAL4 transcriptional
                     activator"
     protein_bind    complement(7998..8022)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    8177..8232
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(8593..8609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8617..8633
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(8641..8671)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8686..8707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    8880..9716
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        complement(9746..9764)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9785..9801)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    9809..9825
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(9833..9863)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(9878..9899)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
ORIGIN
        1 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
       61 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      121 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      181 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      241 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      301 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      361 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      421 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      481 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      541 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      601 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      661 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
      721 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
      781 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
      841 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
      901 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
      961 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1021 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1081 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1141 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1201 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1261 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1321 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1381 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1441 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1501 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1561 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1621 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1681 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     1741 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     1801 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     1861 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     1921 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     1981 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2041 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2101 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2161 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2221 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2281 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2341 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2401 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     2461 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     2521 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     2581 ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtacctgc tttctctgac
     2641 cagcattctc tcccctgggc ctgtgccgct ttctgtctgc agcttgtggc ctgggtcacc
     2701 tctacggctg gcccagatcc ttccctgccg cctccttcag gttccgtctt cctccactcc
     2761 ctcttcccct tgctctctgc tgtgttgctg cccaaggatg ctctttccgg agcacttcct
     2821 tctcggcgct gcaccacgtg atgtcctctg agcggatcct ccccgtgtct gggtcctctc
     2881 cgggcatctc tcctccctca cccaacccca tgccgtcttc actcgctggg ttcccttttc
     2941 cttctccttc tggggcctgt gccatctctc gtttcttagg atggccttct ccgacggatg
     3001 tctcccttgc gtcccgcctc cccttcttgt aggcctgcat catcaccgtt tttctggaca
     3061 accccaaagt accccgtctc cctggcttta gccacctctc catcctcttg ctttctttgc
     3121 ctggacaccc cgttctcctg tggattcggg tcacctctca ctcctttcat ttgggcagct
     3181 cccctacccc ccttacctct ctagtctgtg ctagctcttc cagccccctg tcatggcatc
     3241 ttccaggggt ccgagagctc agctagtctt cttcctccaa cccgggcccc tatgtccact
     3301 tcaggacagc atgtttgctg cctccaggga tcctgtgtcc ccgagctggg accaccttat
     3361 attcccaggg ccggttaatg tggctctggt tctgggtact tttatctgtc ccctccaccc
     3421 cacagtgggg caagcttatt accctgttat ccctaaccgg tttaccgagc tcttattggt
     3481 tttcaaactt cattgactgt gccaagcttc tgacctcttc tcttcctccc acagggcctc
     3541 gagacaattg catcgatggt accagtgaaa gtctgcaatg aattcgagtt ggcgcgcctg
     3601 tcattctaaa tctctctttc agcctaaagc tttttccccg tatcccccca ggtgtctgca
     3661 ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc
     3721 ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg
     3781 agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg
     3841 gctttgggag ggggctgtcc ccgtgagctc ccaggcgcgc ctgtcattct aaatctctct
     3901 ttcagcctaa agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga
     3961 gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac
     4021 gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc
     4081 tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg
     4141 tccccgtgag ctcccaggcg cgccaactcg gtaccgtatc gatgtcgacg ttaacgctag
     4201 tgatatcaac tttgtataga aaagttggct ccgaattcgc ccttattaat tggctccggt
     4261 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttgtg gggaggggtc
     4321 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     4381 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     4441 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
     4501 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
     4561 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
     4621 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
     4681 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     4741 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     4801 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     4861 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     4921 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     4981 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     5041 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     5101 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     5161 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     5221 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     5281 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     5341 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     5401 agtttttttc ttccatttca ggtggatccc aagggcgaat tcgacccaag tttgtacaaa
     5461 aaagcaggct ggatcccggt accctcgaca cctgctgaca ggtcgccacc atggtgtcct
     5521 ggggccggtt catctgcctg gtggtggtca caatggccac cctgagcctg gccagaccca
     5581 gcttcagcct ggtggaagat accaccctgg aaggcggcga gcagaagctg atcagcgaag
     5641 aggacctggg atccctggaa gccgaggcag ctgctaaaga ggctgcagcc aaggccctgg
     5701 aagaattcct gcctgcccct ggcagagaga aagagatcac cgccagcccc gactacctgg
     5761 aaatcgccat ctactgtatc ggcgtgttcc tgatcgcatg catggtggtc accgtgatcc
     5821 tgtgccggat gaagaacacc accaagaagc ccgacttcag cagccagccc gccgtgcaca
     5881 agctgaccaa gagaatcccc ctgcggagac aggtgtccgc cgagagcagc agcagcatga
     5941 acagcaacac ccccctggtc cgcatcacca ccagactgag cagcaccgcc gacaccccta
     6001 tgctggccgg cgtgtccgag tacgagctgc ccgaggaccc caagtgggag ttcccccggg
     6061 acaagcctgc aggcggcgga ggctccggtg gtggctctgg catggtgagc gagctgatta
     6121 aggagaacat gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt
     6181 gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggcgg
     6241 tcgagggcgg ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca
     6301 gcaaaacctt catcaaccac acccagggca tccccgactt ctttaagcag tccttccccg
     6361 agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc
     6421 aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact
     6481 tcccatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc tccaccgaga
     6541 ccctgtaccc cgctgacggc ggcctggaag gcagagccga catggccctg aagctcgtgg
     6601 gcgggggcca cctgatctgc aacttgaaga ccacatacag atccaagaaa cccgctaaga
     6661 acctcaagat gcccggcgtc tactatgtgg acagaagact ggaaagaatc aaggaggccg
     6721 acaaagagac ctacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta
     6781 gcaaactggg gcacaaactt aatggcggag gtggatctgg cggaggtgga tcgggcggag
     6841 gtggatcaac cggtatggcc gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg
     6901 ctgggggctc tctgagactc ttctgtgcgg cctctggatt caccttcggt accagtaata
     6961 tggcctggct ccgccaggct ccagggaagc gtcgcgagtg ggtcgcactt attacgatta
     7021 gtggctacac agactatgca gactccgtga aggaccgatt caccatatcc agagacaacg
     7081 ccaagaacac ggtgtctctg caaatgaaca gcctgaaacc tgaggacacg gccatttatt
     7141 tttgcgcccg gcgtgtaggg tctgagtatg atttgtgggg ccaggggacc caggtcactg
     7201 tctcctcagg cggccgcggc ggaggtggat ctggcggagg tggatcgggc ggaggtggat
     7261 cagagaacct gtacttccag ctgatggccc ccccgaccga tgtcagcctg ggggacgagc
     7321 tccacttaga cggcgaggac gtggcgatgg cgcatgccga cgcgctagac gatttcgatc
     7381 tggacatgtt gggggacggg gattccccgg gtccgggatt taccccccac gactccgccc
     7441 cctacggcgc tctggatatg gccgacttcg agtttgagca gatgtttacc gatgcccttg
     7501 gaattgacga gtacggtggg acgcgtatga agctactgtc ttctatcgaa caagcatgcg
     7561 atatttgccg acttaaaaag ctcaagtgct ccaaagaaaa accgaagtgc gccaagtgtc
     7621 tgaagaacaa ctgggagtgt cgctactctc ccaaaaccaa aaggtctccg ctgactaggg
     7681 cacatctgac agaagtggaa tcaaggctag aaagactgga acagctattt ctactgattt
     7741 ttcctcgaga agaccttgac atgattttga aaatggattc tttacaggat ataaaagcat
     7801 tgttaacagg attatttgta caagataatg tgaataaaga tgccgtcaca gatagattgg
     7861 cttcagtgga gactgatatg cctctaacat tgagacagca tagaataagt gcgacatcat
     7921 catcggaaga gagtagtaac aaaggtcaaa gacagttgac tgtataaaag cttgcttcag
     7981 tgcaggtgaa ttctaccacc cagctttctt gtacaaagtg gtacgcgtga attcactcct
     8041 caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     8101 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     8161 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     8221 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     8281 tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg
     8341 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     8401 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     8461 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     8521 ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt
     8581 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     8641 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     8701 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg
     8761 gatcgacagt actaagcttg gtgcgttttt atgcttgtag tattgtataa tgtttttaag
     8821 atccttaatt aatagggata acagggtaat ctcgaggttt aaacgtcgac gcggccgcta
     8881 ctagggacag gattggtgac agaaaagccc catccttagg cctcctcctt cctagtctcc
     8941 tgatattggg tctaaccccc acctcctgtt aggcagattc cttatctggt gacacacccc
     9001 catttcctgg agccatctct ctccttgcca gaacctctaa ggtttgctta cgatggagcc
     9061 agagaggatc ctgggaggga gagcttggca gggggtggga gggaaggggg ggatgcgtga
     9121 cctgcccggt tctcagtggc caccctgcgc taccctctcc cagaacctga gctgctctga
     9181 cgcggctgtc tggtgcgttt cactgatcct ggtgctgcag cttccttaca cttcccaaga
     9241 ggagaagcag tttggaaaaa caaaatcaga ataagttggt cctgagttct aactttggct
     9301 cttcaccttt ctagtcccca atttatattg ttcctccgtg cgtcagtttt acctgtgaga
     9361 taaggccagt agccagcccc gtcctggcag ggctgtggtg aggagggggg tgtccgtgtg
     9421 gaaaactccc tttgtgagaa tggtgcgtcc taggtgttca ccaggtcgtg gccgcctcta
     9481 ctccctttct ctttctccat ccttctttcc ttaaagagtc cccagtgcta tctgggacat
     9541 attcctccgc ccagagcagg gtcccgcttc cctaaggccc tgctctgggc ttctgggttt
     9601 gagtccttgg caagcccagg agaggcgctc aggcttccct gtcccccttc ctcgtccacc
     9661 atctcatgcc cctggctctc ctgccccttc cctacagggg ttcctggctc tgctctccac
     9721 cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     9781 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     9841 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     9901 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     9961 gaatc
//
