LOCUS       40924_18921        7683 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Mammalian expression vector pEXPR66, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7683)
  AUTHORS   Siciliano V.
  TITLE     Engineering modular intracellular protein sensor-actuator devices
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7683)
  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 7683)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7683)
  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            "Unpublished"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..7683
                     /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)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     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"
     protein_bind    4272..4290
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4307..4325
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4343..4361
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4379..4397
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4414..4432
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4450..4468
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        4480..4518
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     protein_bind    4558..4582
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4615..5316
                     /codon_start=1
                     /label=TEV protease
                     /note="tobacco etch virus protease (Kapust et al., 2001)"
                     /translation="SLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHL
                     FRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQRE
                     ERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVG
                     IHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEP
                     FQPVKEATQLMN"
     CDS             5686..5703
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     protein_bind    complement(5716..5740)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    5895..5950
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6311..6327)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6335..6351
                     /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(6359..6389)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6404..6425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    6598..7434
                     /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(7464..7482)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7503..7519)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7527..7543)
                     /label=lac operator
                     /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(7551..7581)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7596..7617)
                     /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 tgcgttttta tgcttgtagt
     3481 attgtataat gtttttaaga tccaagcttc 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 ccttcaggtc cgaggttcta
     4261 gacgagttta ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat
     4321 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
     4381 cctatcagtg atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt
     4441 cgagtttact ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag
     4501 gcctatataa gcagagctcg tttagtgaac cgtcagatcg caaagggcga attcgaccca
     4561 agtttgtaca aaaaagcagg ctcgccaccg cggccgcatt cgtggccggc catgagcctg
     4621 ttcaagggcc ccagagacta caaccctatc agcagcacca tctgccacct gaccaacgag
     4681 agcgacggcc acaccaccag cctgtacggc atcggcttcg gccccttcat catcaccaac
     4741 aagcacctgt tccggcgaaa caacggcacc ctgctggtgc agagcctgca cggcgtgttc
     4801 aaagtgaaga ataccacaac cctgcagcag cacctgatcg acggccggga catgatcatc
     4861 atccggatgc ccaaggactt cccacccttc ccacagaagc tgaagttcag agagccccag
     4921 cgcgaggaac ggatctgtct ggtcacaaca aacttccaga ccaagagcat gagcagcatg
     4981 gtgtccgata ccagctgcac cttccccagc agcgacggca tcttctggaa gcactggatt
     5041 cagaccaagg acggccagtg cggcagtcca ctggtgtcca cccgggatgg cttcatcgtg
     5101 ggcatccaca gcgccagcaa cttcaccaac accaacaact acttcaccag cgtgcccaag
     5161 aacttcatgg aactgctgac caaccaggaa gcccagcagt gggtctccgg ctggcggctg
     5221 aacgccgatt ctgtgctgtg gggcggacac aaggtgttca tggtgaagcc tgaggaaccc
     5281 ttccagcccg tgaaagaggc cacccagctg atgaacgagc tggtgtacag ccagaagctt
     5341 atgggttctc agcctgtgct gactcagtca ccctcggtct ctgcagcccc ccggcaaagg
     5401 gtcaccatct ccgtgtctgg aagcaactcc aacatcggaa gtaatactgt aaactggatc
     5461 cagcagctcc caggaagggc tcccgagctc ctcatgtacg atgacgatct attggcccca
     5521 ggggtctctg accgattttc tggctctagg tctggcacct cagcctccct gaccatcagt
     5581 gggctccagt ctgaggatga ggctgattat tacgcagcaa catgggatga cagcctgaat
     5641 ggttgggtgt tcggcggagg gaccaaggtc accgtcctat ccggacatca ccatcaccat
     5701 cactgaaccg gttgaaccca gctttcttgt acaaagtggt acgcgtgaat tcactcctca
     5761 ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata
     5821 ccactgagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca
     5881 tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt
     5941 gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta
     6001 tttggtttag agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct
     6061 ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa
     6121 agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac
     6181 atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct cctgactact
     6241 cccagtcata gctgtccctc ttctcttatg gagatccctc gacctgcagc ccaagcttgg
     6301 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     6361 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     6421 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga
     6481 tcgacagtac taagcttggt ctaataccca atctctcgtc ttatccagat gttttatacg
     6541 ccttaattaa tagggataac agggtaatct cgaggtttaa acgtcgacgc ggccgctact
     6601 agggacagga ttggtgacag aaaagcccca tccttaggcc tcctccttcc tagtctcctg
     6661 atattgggtc taacccccac ctcctgttag gcagattcct tatctggtga cacaccccca
     6721 tttcctggag ccatctctct ccttgccaga acctctaagg tttgcttacg atggagccag
     6781 agaggatcct gggagggaga gcttggcagg gggtgggagg gaaggggggg atgcgtgacc
     6841 tgcccggttc tcagtggcca ccctgcgcta ccctctccca gaacctgagc tgctctgacg
     6901 cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct tccttacact tcccaagagg
     6961 agaagcagtt tggaaaaaca aaatcagaat aagttggtcc tgagttctaa ctttggctct
     7021 tcacctttct agtccccaat ttatattgtt cctccgtgcg tcagttttac ctgtgagata
     7081 aggccagtag ccagccccgt cctggcaggg ctgtggtgag gaggggggtg tccgtgtgga
     7141 aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc aggtcgtggc cgcctctact
     7201 ccctttctct ttctccatcc ttctttcctt aaagagtccc cagtgctatc tgggacatat
     7261 tcctccgccc agagcagggt cccgcttccc taaggccctg ctctgggctt ctgggtttga
     7321 gtccttggca agcccaggag aggcgctcag gcttccctgt cccccttcct cgtccaccat
     7381 ctcatgcccc tggctctcct gccccttccc tacaggggtt cctggctctg ctctccaccg
     7441 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     7501 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     7561 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     7621 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     7681 atc
//
