LOCUS       40924_42169        7299 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT1_T1_94bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7299)
  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  JOURNAL   Unpublished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 7299)
  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 7299)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7299)
  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            "Unpublished"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..7299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..95
                     /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    129..188
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    247..267
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             309..326
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             327..1022
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1065..1089)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1244..1299
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1660..1676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1684..1700
                     /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(1708..1738)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1753..1774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    1947..2783
                     /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(2813..2831)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2852..2868)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2876..2892)
                     /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(2900..2930)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2945..2966)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3254..3842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4016..4873)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4874..4978)
                     /label=AmpR promoter
     rep_origin      complement(5004..5459)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5601..5617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5627..5645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5660..6463
                     /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    6542..6567
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    7232..7252
                     /label=attB4
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
ORIGIN
        1 cggagtactg tcctccgagc ggagtactgt cctccgagcg gagtactgtc ctccgagcgg
       61 agtactgtcc tccgagcgga gttctgtcct ccgagcggag actctagata cgactcacta
      121 tagaattcta ggcgtgtacg gtgggaggcc tatataagca gagctcgttt agtgaaccgt
      181 cagatcgcct cgagtaggtt gtgtcgcgag tgttggtacg actcactata ggatccaatt
      241 cgaccccaag tttgtacaaa aaagcaggga tcccggtacc ctcgacacct gctgacggag
      301 ccaccatgca ccatcaccat caccatatgg tgagcgagct gattaaggag aacatgcaca
      361 tgaagctgta catggagggc accgtgaaca accaccactt caagtgcaca tccgagggcg
      421 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggcggtcgag ggcggccctc
      481 tccccttcgc cttcgacatc ctggctacca gcttcatgta cggcagcaaa accttcatca
      541 accacaccca gggcatcccc gacttcttta agcagtcctt ccccgagggc ttcacatggg
      601 agagagtcac cacatacgaa gatgggggcg tgctgaccgc tacccaggac accagcctcc
      661 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttccca tccaacggcc
      721 ctgtgatgca gaagaaaaca ctcggctggg aggcctccac cgagacactg taccccgctg
      781 acggcggcct ggaaggcaga gccgacatgg ccctgaagct cgtgggcggg ggccacctga
      841 tctgcaacct taagaccaca tacagatcca agaaacccgc taagaacctc aagatgcccg
      901 gcgtctacta tgtggacagg agactggaaa gaatcaagga ggccgacaaa gagacatacg
      961 tcgagcagca cgaggtggct gtggccagat actgcgacct ccctagcaaa ctggggcaca
     1021 aacttaattc ctagtaacgc tcagtgcagg tgaattctac caccacccag ctttcttgta
     1081 caaagtggta cgcgtgaatt cactcctcag gtgcaggctg cctatcagaa ggtggtggct
     1141 ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat
     1201 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     1261 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     1321 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     1381 atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc
     1441 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     1501 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     1561 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg
     1621 agatccctcg acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     1681 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     1741 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     1801 ccagtcggga aacctgtcgt gccagcggat cgacagtact aagcttggtg aattccctta
     1861 tgtgagtgta aaaggcaggc gagtttgtcc cttaattaat agggataaca gggtaatctc
     1921 gaggtttaaa cgtcgacgcg gccgctacta gggacaggat tggtgacaga aaagccccat
     1981 ccttaggcct cctccttcct agtctcctga tattgggtct aacccccacc tcctgttagg
     2041 cagattcctt atctggtgac acacccccat ttcctggagc catctctctc cttgccagaa
     2101 cctctaaggt ttgcttacga tggagccaga gaggatcctg ggagggagag cttggcaggg
     2161 ggtgggaggg aaggggggga tgcgtgacct gcccggttct cagtggccac cctgcgctac
     2221 cctctcccag aacctgagct gctctgacgc ggctgtctgg tgcgtttcac tgatcctggt
     2281 gctgcagctt ccttacactt cccaagagga gaagcagttt ggaaaaacaa aatcagaata
     2341 agttggtcct gagttctaac tttggctctt cacctttcta gtccccaatt tatattgttc
     2401 ctccgtgcgt cagttttacc tgtgagataa ggccagtagc cagccccgtc ctggcagggc
     2461 tgtggtgagg aggggggtgt ccgtgtggaa aactcccttt gtgagaatgg tgcgtcctag
     2521 gtgttcacca ggtcgtggcc gcctctactc cctttctctt tctccatcct tctttcctta
     2581 aagagtcccc agtgctatct gggacatatt cctccgccca gagcagggtc ccgcttccct
     2641 aaggccctgc tctgggcttc tgggtttgag tccttggcaa gcccaggaga ggcgctcagg
     2701 cttccctgtc ccccttcctc gtccaccatc tcatgcccct ggctctcctg ccccttccct
     2761 acaggggttc ctggctctgc tctccaccgc ggtggagctc cagcttttgt tccctttagt
     2821 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2881 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2941 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3001 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3061 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3121 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3181 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3241 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3301 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3361 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3421 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3481 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3541 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3601 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3661 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3721 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3781 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3841 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3901 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3961 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4021 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4081 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4141 caatgatacc gcgagagcca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4201 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4261 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4321 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4381 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4441 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4501 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4561 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4621 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4681 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4741 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4801 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4861 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4921 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4981 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     5041 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5101 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5161 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     5221 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     5281 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     5341 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     5401 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     5461 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     5521 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     5581 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     5641 atagggcgaa ttgggtacct gctttctctg accagcattc tctcccctgg gcctgtgccg
     5701 ctttctgtct gcagcttgtg gcctgggtca cctctacggc tggcccagat ccttccctgc
     5761 cgcctccttc aggttccgtc ttcctccact ccctcttccc cttgctctct gctgtgttgc
     5821 tgcccaagga tgctctttcc ggagcacttc cttctcggcg ctgcaccacg tgatgtcctc
     5881 tgagcggatc ctccccgtgt ctgggtcctc tccgggcatc tctcctccct cacccaaccc
     5941 catgccgtct tcactcgctg ggttcccttt tccttctcct tctggggcct gtgccatctc
     6001 tcgtttctta ggatggcctt ctccgacgga tgtctccctt gcgtcccgcc tccccttctt
     6061 gtaggcctgc atcatcaccg tttttctgga caaccccaaa gtaccccgtc tccctggctt
     6121 tagccacctc tccatcctct tgctttcttt gcctggacac cccgttctcc tgtggattcg
     6181 ggtcacctct cactcctttc atttgggcag ctcccctacc ccccttacct ctctagtctg
     6241 tgctagctct tccagccccc tgtcatggca tcttccaggg gtccgagagc tcagctagtc
     6301 ttcttcctcc aacccgggcc cctatgtcca cttcaggaca gcatgtttgc tgcctccagg
     6361 gatcctgtgt ccccgagctg ggaccacctt atattcccag ggccggttaa tgtggctctg
     6421 gttctgggta cttttatctg tcccctccac cccacagtgg ggcaagctta ttaccctgtt
     6481 atccctaacc ggtctaatac ccaatctctc gtcttatcca gatgttttat acgccaagct
     6541 tctgacctct tctcttcctc ccacagggcc tcgagacaat tgattaacat cgatacggta
     6601 ccgagttggc gcgcctggga gctcacgggg acagcccccc cccaaagccc ccagggatgt
     6661 aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc
     6721 ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg
     6781 tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg
     6841 gggggatacg gggaaaaagc tttaggctga aagagagatt tagaatgaca ggcgcgcctg
     6901 gccatacatc gatacggtac cgagttggcg cgcctgggag ctcacgggga cagccccccc
     6961 ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc
     7021 cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac
     7081 agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct
     7141 ttgagcctgc agacacctgg ggggatacgg ggaaaaagct ttaggctgaa agagagattt
     7201 agaatgacag gcgcgcctgg tggcgaacga tcaactttgt atagaaaagt tggctccgaa
     7261 tttctcgaca gatctcatgt gattacgcca agctacggg
//
