LOCUS       40924_18866        8137 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Mammalian expression vector pEXPR21, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8137)
  AUTHORS   Siciliano V.
  TITLE     Engineering modular intracellular protein sensor-actuator devices
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8137)
  AUTHORS   Siciliano V.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-MAR-2018) Biological Engineering, Massachusetts 
            Institute of Technology, 500 Tech Square, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 8137)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8137)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (22-MAR-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..8137
                     /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"
     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"
     regulatory      4616..4625
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4634..4957
                     /label=DD
                     /note="destabilization domain that can be stabilized by
                     Shield1 in the ProteoTuner(TM) system"
     misc_feature    4958..5362
                     /label=Region: scFv162
                     /note="Region: scFv162"
     CDS             5423..6124
                     /label=TEV protease
                     /note="tobacco etch virus protease (Kapust et al., 2001)"
     protein_bind    complement(6170..6194)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    6349..6404
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6765..6781)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6789..6805
                     /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(6813..6843)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6858..6879
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    7052..7888
                     /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(7918..7936)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7957..7973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7981..7997)
                     /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(8005..8035)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(8050..8071)
                     /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 ctggatcccg gtaccctcga cacctgctga caggtgccac
     4621 catgggcggc cgcatgggag tgcaggtgga gaccatcagc ccaggagatg gcaggacatt
     4681 ccccaagcgc gggcagactt gcgtggtgca ctacaccggc atgctggagg acgggaagaa
     4741 agtggatagc tccagggacc gcaacaagcc cttcaagttc atgctgggaa agcaggaagt
     4801 gatccgcggg tgggaggaag gagtggccca gatgagtgtg ggccagcggg ctaaactgac
     4861 tatttcacct gactacgcct atggagctac cggccaccca gggatcattc cccctcatgc
     4921 caccctggtg ttcgatgtgg agctgctgaa gccagaaatg gccgaggtcc aggtgcagca
     4981 gtctggggca gagcttgtga ggtcaggggc ctcagtcaag ttgtcctgca cagcttctgg
     5041 cttcaacatt aaagactact atatgcactg ggtgaagcag aggcctgaac agggcctgga
     5101 gtggattgga tggattgatc ctgcgaatgg taatactaaa tatgacccga agttccaggg
     5161 caaggccact ataacagcag acacatcctc caacacagcc tacctgcagc tcagcagcct
     5221 gacatctgag gacactgccg tctattactg tgctagatgg gactggtact tcgatgtctg
     5281 gggcgcaggg accacagtca cagtctcctc gggaggtggt ggatccggcg gtggcggttc
     5341 tggtggaggt ggatctgtgt ttcctgcagg aagtggcgga ggtggatctg gcggaggtgg
     5401 atcgggcgga ggtggatcag gcagcctgtt caagggcccc agagactaca accctatcag
     5461 cagcaccatc tgccacctga ccaacgagag cgacggccac accaccagcc tgtacggcat
     5521 cggcttcggc cccttcatca tcaccaacaa gcacctgttc cggcgaaaca acggcaccct
     5581 gctggtgcag agcctgcacg gcgtgttcaa agtgaagaat accacaaccc tgcagcagca
     5641 cctgatcgac ggccgggaca tgatcatcat ccggatgccc aaggacttcc cacccttccc
     5701 acagaagctg aagttcagag agccccagcg cgaggaacgg atctgtctgg tcacaacaaa
     5761 cttccagacc aagagcatga gcagcatggt gtccgatacc agctgcacct tccccagcag
     5821 cgacggcatc ttctggaagc actggattca gaccaaggac ggccagtgcg gcagtccact
     5881 ggtgtccacc cgggatggct tcatcgtggg catccacagc gccagcaact tcaccaacac
     5941 caacaactac ttcaccagcg tgcccaagaa cttcatggaa ctgctgacca accaggaagc
     6001 ccagcagtgg gtctccggct ggcggctgaa cgccgattct gtgctgtggg gcggacacaa
     6061 ggtgttcatg gtgaagcctg aggaaccctt ccagcccgtg aaagaggcca cccagctgat
     6121 gaacgagctg gtgtacagcc agtgagcttc agtgcaggtg aattctacca cccagctttc
     6181 ttgtacaaag tggtacgcgt gaattcactc ctcaggtgca ggctgcctat cagaaggtgg
     6241 tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca
     6301 aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat
     6361 ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg
     6421 ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg
     6481 cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac
     6541 agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt
     6601 ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt
     6661 tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct
     6721 tatggagatc cctcgacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct
     6781 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     6841 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     6901 gctttccagt cgggaaacct gtcgtgccag cggatcgaca gtactaagct tggtctaata
     6961 cccaatctct cgtcttatcc agatgtttta tacgccttaa ttaataggga taacagggta
     7021 atctcgaggt ttaaacgtcg acgcggccgc tactagggac aggattggtg acagaaaagc
     7081 cccatcctta ggcctcctcc ttcctagtct cctgatattg ggtctaaccc ccacctcctg
     7141 ttaggcagat tccttatctg gtgacacacc cccatttcct ggagccatct ctctccttgc
     7201 cagaacctct aaggtttgct tacgatggag ccagagagga tcctgggagg gagagcttgg
     7261 cagggggtgg gagggaaggg ggggatgcgt gacctgcccg gttctcagtg gccaccctgc
     7321 gctaccctct cccagaacct gagctgctct gacgcggctg tctggtgcgt ttcactgatc
     7381 ctggtgctgc agcttcctta cacttcccaa gaggagaagc agtttggaaa aacaaaatca
     7441 gaataagttg gtcctgagtt ctaactttgg ctcttcacct ttctagtccc caatttatat
     7501 tgttcctccg tgcgtcagtt ttacctgtga gataaggcca gtagccagcc ccgtcctggc
     7561 agggctgtgg tgaggagggg ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt
     7621 cctaggtgtt caccaggtcg tggccgcctc tactcccttt ctctttctcc atccttcttt
     7681 ccttaaagag tccccagtgc tatctgggac atattcctcc gcccagagca gggtcccgct
     7741 tccctaaggc cctgctctgg gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc
     7801 tcaggcttcc ctgtccccct tcctcgtcca ccatctcatg cccctggctc tcctgcccct
     7861 tccctacagg ggttcctggc tctgctctcc accgcggtgg agctccagct tttgttccct
     7921 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     7981 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     8041 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     8101 gtcgggaaac ctgtcgtgcc agctgcatta atgaatc
//
