LOCUS       40924_18926        8047 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Mammalian expression vector pEXPR71, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8047)
  AUTHORS   Siciliano V.
  TITLE     Engineering modular intracellular protein sensor-actuator devices
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8047)
  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 8047)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8047)
  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            "Unpublished"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..8047
                     /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             4598..4921
                     /codon_start=1
                     /label=DD
                     /note="destabilization domain that can be stabilized by
                     Shield1 in the ProteoTuner(TM) system"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     CDS             4934..5635
                     /codon_start=1
                     /label=TEV protease
                     /note="tobacco etch virus protease (Kapust et al., 2001)"
                     /translation="SLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHL
                     FRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQRE
                     ERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVG
                     IHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEP
                     FQPVKEATQLMN"
     CDS             6050..6067
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     protein_bind    complement(6080..6104)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    6259..6314
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6675..6691)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6699..6715
                     /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(6723..6753)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6768..6789
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    6962..7798
                     /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(7828..7846)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7867..7883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7891..7907)
                     /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(7915..7945)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7960..7981)
                     /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 cggccgcatg ggagtgcagg tggagaccat
     4621 cagcccagga gatggcagga cattccccaa gcgcgggcag acttgcgtgg tgcactacac
     4681 cggcatgctg gaggacggga agaaagtgga tagctccagg gaccgcaaca agcccttcaa
     4741 gttcatgctg ggaaagcagg aagtgatccg cgggtgggag gaaggagtgg cccagatgag
     4801 tgtgggccag cgggctaaac tgactatttc acctgactac gcctatggag ctaccggcca
     4861 cccagggatc attccccctc atgccaccct ggtgttcgat gtggagctgc tgaagccaga
     4921 aggccggcct atgagcctgt tcaagggccc cagagactac aaccctatca gcagcaccat
     4981 ctgccacctg accaacgaga gcgacggcca caccaccagc ctgtacggca tcggcttcgg
     5041 ccccttcatc atcaccaaca agcacctgtt ccggcgaaac aacggcaccc tgctggtgca
     5101 gagcctgcac ggcgtgttca aagtgaagaa taccacaacc ctgcagcagc acctgatcga
     5161 cggccgggac atgatcatca tccggatgcc caaggacttc ccacccttcc cacagaagct
     5221 gaagttcaga gagccccagc gcgaggaacg gatctgtctg gtcacaacaa acttccagac
     5281 caagagcatg agcagcatgg tgtccgatac cagctgcacc ttccccagca gcgacggcat
     5341 cttctggaag cactggattc agaccaagga cggccagtgc ggcagtccac tggtgtccac
     5401 ccgggatggc ttcatcgtgg gcatccacag cgccagcaac ttcaccaaca ccaacaacta
     5461 cttcaccagc gtgcccaaga acttcatgga actgctgacc aaccaggaag cccagcagtg
     5521 ggtctccggc tggcggctga acgccgattc tgtgctgtgg ggcggacaca aggtgttcat
     5581 ggtgaagcct gaggaaccct tccagcccgt gaaagaggcc acccagctga tgaacgagct
     5641 ggtgtacagc cagggcggag gtggatctgg cggaggtgga tcgggcggag gtggatcaaa
     5701 gcttatgggt tctcagcctg tgctgactca gtcaccctcg gtctctgcag ccccccggca
     5761 aagggtcacc atctccgtgt ctggaagcaa ctccaacatc ggaagtaata ctgtaaactg
     5821 gatccagcag ctcccaggaa gggctcccga gctcctcatg tacgatgacg atctattggc
     5881 cccaggggtc tctgaccgat tttctggctc taggtctggc acctcagcct ccctgaccat
     5941 cagtgggctc cagtctgagg atgaggctga ttattacgca gcaacatggg atgacagcct
     6001 gaatggttgg gtgttcggcg gagggaccaa ggtcaccgtc ctatccggac atcaccatca
     6061 ccatcactga accggttgaa cccagctttc ttgtacaaag tggtacgcgt gaattcactc
     6121 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca
     6181 aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg
     6241 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt
     6301 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg
     6361 agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt
     6421 ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata
     6481 gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt
     6541 taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac
     6601 tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc
     6661 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     6721 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     6781 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6841 cggatcgaca gtactaagct tggtctaata cccaatctct cgtcttatcc agatgtttta
     6901 tacgccttaa ttaataggga taacagggta atctcgaggt ttaaacgtcg acgcggccgc
     6961 tactagggac aggattggtg acagaaaagc cccatcctta ggcctcctcc ttcctagtct
     7021 cctgatattg ggtctaaccc ccacctcctg ttaggcagat tccttatctg gtgacacacc
     7081 cccatttcct ggagccatct ctctccttgc cagaacctct aaggtttgct tacgatggag
     7141 ccagagagga tcctgggagg gagagcttgg cagggggtgg gagggaaggg ggggatgcgt
     7201 gacctgcccg gttctcagtg gccaccctgc gctaccctct cccagaacct gagctgctct
     7261 gacgcggctg tctggtgcgt ttcactgatc ctggtgctgc agcttcctta cacttcccaa
     7321 gaggagaagc agtttggaaa aacaaaatca gaataagttg gtcctgagtt ctaactttgg
     7381 ctcttcacct ttctagtccc caatttatat tgttcctccg tgcgtcagtt ttacctgtga
     7441 gataaggcca gtagccagcc ccgtcctggc agggctgtgg tgaggagggg ggtgtccgtg
     7501 tggaaaactc cctttgtgag aatggtgcgt cctaggtgtt caccaggtcg tggccgcctc
     7561 tactcccttt ctctttctcc atccttcttt ccttaaagag tccccagtgc tatctgggac
     7621 atattcctcc gcccagagca gggtcccgct tccctaaggc cctgctctgg gcttctgggt
     7681 ttgagtcctt ggcaagccca ggagaggcgc tcaggcttcc ctgtccccct tcctcgtcca
     7741 ccatctcatg cccctggctc tcctgcccct tccctacagg ggttcctggc tctgctctcc
     7801 accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     7861 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     7921 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     7981 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     8041 atgaatc
//
