LOCUS       40924_48343        7317 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pZD_2-3-T14+T14x3+72-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7317)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z.
  TITLE     Modular construction of mammalian gene circuits using TALE 
            transcriptional repressors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7317)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-JAN-2015) Bioinformatics Division/Center for Synthetic
REFERENCE   3  (bases 1 to 7317)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7317)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (08-JAN-2015) Bioinformatics Division/Center for Synthetic "
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4..861)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(862..966)
                     /label=AmpR promoter
     rep_origin      complement(992..1447)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     1589..1605
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1615..1633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1648..2451
                     /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    2530..2555
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    3220..3240
                     /label=attB4
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     protein_bind    3288..3382
                     /label=5X UAS
                     /note="five tandem copies of the 'ScaI site' 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     misc_feature    3396..3412
                     /label=TALER14 bs
                     /note="TALER14 bs"
     misc_feature    3419..3478
                     /label=miniCMV
                     /note="miniCMV"
     misc_feature    3485..3501
                     /label=TALER14 bs
                     /note="TALER14 bs"
     misc_feature    3502..3518
                     /label=TALER14 bs
                     /note="TALER14 bs"
     misc_feature    3519..3535
                     /label=TALER14 bs
                     /note="TALER14 bs"
     protein_bind    3552..3572
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             3614..3631
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             3632..4327
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(4370..4394)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4549..4604
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4965..4981)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4989..5005
                     /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(5013..5043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5058..5079
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    5252..6088
                     /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(6118..6136)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6157..6173)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6181..6197)
                     /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(6205..6235)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6250..6271)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6559..7147)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     1021 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     1081 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     1141 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     1201 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     1261 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     1321 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     1381 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     1441 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     1501 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     1561 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
     1621 gactcactat agggcgaatt gggtacctgc tttctctgac cagcattctc tcccctgggc
     1681 ctgtgccgct ttctgtctgc agcttgtggc ctgggtcacc tctacggctg gcccagatcc
     1741 ttccctgccg cctccttcag gttccgtctt cctccactcc ctcttcccct tgctctctgc
     1801 tgtgttgctg cccaaggatg ctctttccgg agcacttcct tctcggcgct gcaccacgtg
     1861 atgtcctctg agcggatcct ccccgtgtct gggtcctctc cgggcatctc tcctccctca
     1921 cccaacccca tgccgtcttc actcgctggg ttcccttttc cttctccttc tggggcctgt
     1981 gccatctctc gtttcttagg atggccttct ccgacggatg tctcccttgc gtcccgcctc
     2041 cccttcttgt aggcctgcat catcaccgtt tttctggaca accccaaagt accccgtctc
     2101 cctggcttta gccacctctc catcctcttg ctttctttgc ctggacaccc cgttctcctg
     2161 tggattcggg tcacctctca ctcctttcat ttgggcagct cccctacccc ccttacctct
     2221 ctagtctgtg ctagctcttc cagccccctg tcatggcatc ttccaggggt ccgagagctc
     2281 agctagtctt cttcctccaa cccgggcccc tatgtccact tcaggacagc atgtttgctg
     2341 cctccaggga tcctgtgtcc ccgagctggg accaccttat attcccaggg ccggttaatg
     2401 tggctctggt tctgggtact tttatctgtc ccctccaccc cacagtgggg caagcttatt
     2461 accctgttat ccctaaccgg tgcgttttta tgcttgtagt attgtataat gtttttaaga
     2521 tccaagcttc tgacctcttc tcttcctccc acagggcctc gagacaattg attaacatcg
     2581 atacggtacc gagttggcgc gcctgggagc tcacggggac agcccccccc caaagccccc
     2641 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc
     2701 tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac
     2761 ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca
     2821 gacacctggg gggatacggg gaaaaagctt taggctgaaa gagagattta gaatgacagg
     2881 cgcgcctggc catacatcga tacggtaccg agttggcgcg cctgggagct cacggggaca
     2941 gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc
     3001 gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt
     3061 gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc
     3121 gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt aggctgaaag
     3181 agagatttag aatgacaggc gcgcctggtg gcgaacgatc aactttgtat agaaaagttg
     3241 gctccgaatt tctcgacaga tctcatgtga ttacgccaag ctacgggcgg agtactgtcc
     3301 tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt actgtcctcc
     3361 gagcggagtt ctgtcctccg agcggagact ctagataccc ctctccgctt ctgaattcta
     3421 ggcgtgtacg gtgggaggcc tatataagca gagctcgttt agtgaaccgt cagatcgcct
     3481 cgagtacccc tctccgcttc ttacccctct ccgcttctta cccctctccg cttctggatc
     3541 caattcgacc ccaagtttgt acaaaaaagc agggatcccg gtaccctcga cacctgctga
     3601 cggagccacc atgcaccatc accatcacca tatggtgagc gagctgatta aggagaacat
     3661 gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga
     3721 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggcgg tcgagggcgg
     3781 ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca gcaaaacctt
     3841 catcaaccac acccagggca tccccgactt ctttaagcag tccttccccg agggcttcac
     3901 atgggagaga gtcaccacat acgaagatgg gggcgtgctg accgctaccc aggacaccag
     3961 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa
     4021 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc tccaccgaga cactgtaccc
     4081 cgctgacggc ggcctggaag gcagagccga catggccctg aagctcgtgg gcgggggcca
     4141 cctgatctgc aaccttaaga ccacatacag atccaagaaa cccgctaaga acctcaagat
     4201 gcccggcgtc tactatgtgg acaggagact ggaaagaatc aaggaggccg acaaagagac
     4261 atacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg
     4321 gcacaaactt aattcctagt aacgctcagt gcaggtgaat tctaccacca cccagctttc
     4381 ttgtacaaag tggtacgcgt gaattcactc ctcaggtgca ggctgcctat cagaaggtgg
     4441 tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca
     4501 aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat
     4561 ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg
     4621 ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg
     4681 cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac
     4741 agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt
     4801 ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt
     4861 tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct
     4921 tatggagatc cctcgacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct
     4981 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5041 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5101 gctttccagt cgggaaacct gtcgtgccag cggatcgaca gtactaagct tggtctaata
     5161 cccaatctct cgtcttatcc agatgtttta tacgccttaa ttaataggga taacagggta
     5221 atctcgaggt ttaaacgtcg acgcggccgc tactagggac aggattggtg acagaaaagc
     5281 cccatcctta ggcctcctcc ttcctagtct cctgatattg ggtctaaccc ccacctcctg
     5341 ttaggcagat tccttatctg gtgacacacc cccatttcct ggagccatct ctctccttgc
     5401 cagaacctct aaggtttgct tacgatggag ccagagagga tcctgggagg gagagcttgg
     5461 cagggggtgg gagggaaggg ggggatgcgt gacctgcccg gttctcagtg gccaccctgc
     5521 gctaccctct cccagaacct gagctgctct gacgcggctg tctggtgcgt ttcactgatc
     5581 ctggtgctgc agcttcctta cacttcccaa gaggagaagc agtttggaaa aacaaaatca
     5641 gaataagttg gtcctgagtt ctaactttgg ctcttcacct ttctagtccc caatttatat
     5701 tgttcctccg tgcgtcagtt ttacctgtga gataaggcca gtagccagcc ccgtcctggc
     5761 agggctgtgg tgaggagggg ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt
     5821 cctaggtgtt caccaggtcg tggccgcctc tactcccttt ctctttctcc atccttcttt
     5881 ccttaaagag tccccagtgc tatctgggac atattcctcc gcccagagca gggtcccgct
     5941 tccctaaggc cctgctctgg gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc
     6001 tcaggcttcc ctgtccccct tcctcgtcca ccatctcatg cccctggctc tcctgcccct
     6061 tccctacagg ggttcctggc tctgctctcc accgcggtgg agctccagct tttgttccct
     6121 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     6181 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     6241 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     6301 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6361 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     6421 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6481 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6541 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6601 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6661 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6721 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6781 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6841 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6901 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6961 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     7021 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     7081 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     7141 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     7201 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     7261 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacag
//
