LOCUS       40924_42324        7321 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT5_T5x3_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7321)
  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  JOURNAL   Unpublished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 7321)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-SEP-2014) Bioinformatics Division/Center for Synthetic
REFERENCE   3  (bases 1 to 7321)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7321)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (03-SEP-2014) 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..7321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..21
                     /label=attB4
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     protein_bind    69..163
                     /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)"
     protein_bind    177..194
                     /label=TALER5 bs binding site
                     /bound_moiety="TALER5 bs"
     misc_feature    201..260
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    267..284
                     /label=TALER5 bs binding site
                     /bound_moiety="TALER5 bs"
     protein_bind    285..302
                     /label=TALER5 bs binding site
                     /bound_moiety="TALER5 bs"
     protein_bind    303..320
                     /label=TALER5 bs binding site
                     /bound_moiety="TALER5 bs"
     protein_bind    337..357
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             399..416
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             417..1112
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1155..1179)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1334..1389
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1750..1766)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1774..1790
                     /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(1798..1828)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1843..1864
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2037..2873
                     /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(2903..2921)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2942..2958)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2966..2982)
                     /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(2990..3020)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3035..3056)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3344..3932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4106..4963)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4964..5068)
                     /label=AmpR promoter
     rep_origin      complement(5094..5549)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5691..5707
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5717..5735
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5750..6553
                     /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    6632..6657
                     /label=SA
                     /note="splice acceptor site"
ORIGIN
        1 caactttgta tagaaaagtt ggctccgaat ttctcgacag atctcatgtg attacgccaa
       61 gctacgggcg gagtactgtc ctccgagcgg agtactgtcc tccgagcgga gtactgtcct
      121 ccgagcggag tactgtcctc cgagcggagt tctgtcctcc gagcggagac tctagatact
      181 gaataatcaa cactgaattc taggcgtgta cggtgggagg cctatataag cagagctcgt
      241 ttagtgaacc gtcagatcgc ctcgagtact gaataatcaa cacttactga ataatcaaca
      301 cttactgaat aatcaacact ggatccaatt cgaccccaag tttgtacaaa aaagcaggga
      361 tcccggtacc ctcgacacct gctgacggag ccaccatgca ccatcaccat caccatatgg
      421 tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc accgtgaaca
      481 accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca
      541 tgagaatcaa ggcggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacca
      601 gcttcatgta cggcagcaaa accttcatca accacaccca gggcatcccc gacttcttta
      661 agcagtcctt ccccgagggc ttcacatggg agagagtcac cacatacgaa gatgggggcg
      721 tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga
      781 tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg
      841 aggcctccac cgagacactg taccccgctg acggcggcct ggaaggcaga gccgacatgg
      901 ccctgaagct cgtgggcggg ggccacctga tctgcaacct taagaccaca tacagatcca
      961 agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggacagg agactggaaa
     1021 gaatcaagga ggccgacaaa gagacatacg tcgagcagca cgaggtggct gtggccagat
     1081 actgcgacct ccctagcaaa ctggggcaca aacttaattc ctagtaacgc tcagtgcagg
     1141 tgaattctac caccacccag ctttcttgta caaagtggta cgcgtgaatt cactcctcag
     1201 gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac
     1261 cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat
     1321 ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg
     1381 tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat
     1441 ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta
     1501 taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa
     1561 gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca
     1621 tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc
     1681 ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttggc
     1741 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1801 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1861 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
     1921 cgacagtact aagcttggtc taatacccaa tctctcgtct tatccagatg ttttatacgc
     1981 cttaattaat agggataaca gggtaatctc gaggtttaaa cgtcgacgcg gccgctacta
     2041 gggacaggat tggtgacaga aaagccccat ccttaggcct cctccttcct agtctcctga
     2101 tattgggtct aacccccacc tcctgttagg cagattcctt atctggtgac acacccccat
     2161 ttcctggagc catctctctc cttgccagaa cctctaaggt ttgcttacga tggagccaga
     2221 gaggatcctg ggagggagag cttggcaggg ggtgggaggg aaggggggga tgcgtgacct
     2281 gcccggttct cagtggccac cctgcgctac cctctcccag aacctgagct gctctgacgc
     2341 ggctgtctgg tgcgtttcac tgatcctggt gctgcagctt ccttacactt cccaagagga
     2401 gaagcagttt ggaaaaacaa aatcagaata agttggtcct gagttctaac tttggctctt
     2461 cacctttcta gtccccaatt tatattgttc ctccgtgcgt cagttttacc tgtgagataa
     2521 ggccagtagc cagccccgtc ctggcagggc tgtggtgagg aggggggtgt ccgtgtggaa
     2581 aactcccttt gtgagaatgg tgcgtcctag gtgttcacca ggtcgtggcc gcctctactc
     2641 cctttctctt tctccatcct tctttcctta aagagtcccc agtgctatct gggacatatt
     2701 cctccgccca gagcagggtc ccgcttccct aaggccctgc tctgggcttc tgggtttgag
     2761 tccttggcaa gcccaggaga ggcgctcagg cttccctgtc ccccttcctc gtccaccatc
     2821 tcatgcccct ggctctcctg ccccttccct acaggggttc ctggctctgc tctccaccgc
     2881 ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     2941 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3001 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3061 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3121 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3181 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3241 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3301 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3361 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3421 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3481 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3541 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3601 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3661 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3721 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3781 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3841 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3901 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4021 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4081 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4141 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4201 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4261 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4321 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4381 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4441 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4501 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4561 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4621 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4681 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4741 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4801 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4861 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4921 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5101 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5161 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5221 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5281 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5341 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5401 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5461 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5521 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     5581 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     5641 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     5701 ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtacct gctttctctg
     5761 accagcattc tctcccctgg gcctgtgccg ctttctgtct gcagcttgtg gcctgggtca
     5821 cctctacggc tggcccagat ccttccctgc cgcctccttc aggttccgtc ttcctccact
     5881 ccctcttccc cttgctctct gctgtgttgc tgcccaagga tgctctttcc ggagcacttc
     5941 cttctcggcg ctgcaccacg tgatgtcctc tgagcggatc ctccccgtgt ctgggtcctc
     6001 tccgggcatc tctcctccct cacccaaccc catgccgtct tcactcgctg ggttcccttt
     6061 tccttctcct tctggggcct gtgccatctc tcgtttctta ggatggcctt ctccgacgga
     6121 tgtctccctt gcgtcccgcc tccccttctt gtaggcctgc atcatcaccg tttttctgga
     6181 caaccccaaa gtaccccgtc tccctggctt tagccacctc tccatcctct tgctttcttt
     6241 gcctggacac cccgttctcc tgtggattcg ggtcacctct cactcctttc atttgggcag
     6301 ctcccctacc ccccttacct ctctagtctg tgctagctct tccagccccc tgtcatggca
     6361 tcttccaggg gtccgagagc tcagctagtc ttcttcctcc aacccgggcc cctatgtcca
     6421 cttcaggaca gcatgtttgc tgcctccagg gatcctgtgt ccccgagctg ggaccacctt
     6481 atattcccag ggccggttaa tgtggctctg gttctgggta cttttatctg tcccctccac
     6541 cccacagtgg ggcaagctta ttaccctgtt atccctaacc ggtgcgtttt tatgcttgta
     6601 gtattgtata atgtttttaa gatccaagct tctgacctct tctcttcctc ccacagggcc
     6661 tcgagacaat tgattaacat cgatacggta ccgagttggc gcgcctggga gctcacgggg
     6721 acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc
     6781 agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag
     6841 cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt
     6901 ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaagc tttaggctga
     6961 aagagagatt tagaatgaca ggcgcgcctg gccatacatc gatacggtac cgagttggcg
     7021 cgcctgggag ctcacgggga cagccccccc ccaaagcccc cagggatgta attacgtccc
     7081 tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc
     7141 cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat
     7201 cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg
     7261 ggaaaaagct ttaggctgaa agagagattt agaatgacag gcgcgcctgg tggcgaacga
     7321 t
//
