LOCUS       40924_42279        7275 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT31_T31_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7275)
  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  JOURNAL   Unpublished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 7275)
  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 7275)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7275)
  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            "Unpublished"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..7275
                     /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..189
                     /label=TALER31 bs binding site
                     /bound_moiety="TALER31 bs"
     misc_feature    196..255
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    262..274
                     /label=TALER31 bs binding site
                     /bound_moiety="TALER31 bs"
     protein_bind    291..311
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             353..370
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             371..1066
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1109..1133)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1288..1343
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1704..1720)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1728..1744
                     /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(1752..1782)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1797..1818
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    1991..2827
                     /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(2857..2875)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2896..2912)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2920..2936)
                     /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(2944..2974)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2989..3010)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3298..3886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4060..4917)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4918..5022)
                     /label=AmpR promoter
     rep_origin      complement(5048..5503)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5645..5661
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5671..5689
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5704..6507
                     /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    6586..6611
                     /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 tctagatagg
      181 agtgagtatg aattctaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt
      241 gaaccgtcag atcgcctcga gtaggagtga gtatggatcc aattcgaccc caagtttgta
      301 caaaaaagca gggatcccgg taccctcgac acctgctgac ggagccacca tgcaccatca
      361 ccatcaccat atggtgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
      421 gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga
      481 gggcacccag accatgagaa tcaaggcggt cgagggcggc cctctcccct tcgccttcga
      541 catcctggct accagcttca tgtacggcag caaaaccttc atcaaccaca cccagggcat
      601 ccccgacttc tttaagcagt ccttccccga gggcttcaca tgggagagag tcaccacata
      661 cgaagatggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
      721 ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa
      781 aacactcggc tgggaggcct ccaccgagac actgtacccc gctgacggcg gcctggaagg
      841 cagagccgac atggccctga agctcgtggg cgggggccac ctgatctgca accttaagac
      901 cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga
      961 caggagactg gaaagaatca aggaggccga caaagagaca tacgtcgagc agcacgaggt
     1021 ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta attcctagta
     1081 acgctcagtg caggtgaatt ctaccaccac ccagctttct tgtacaaagt ggtacgcgtg
     1141 aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     1201 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     1261 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     1321 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     1381 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     1441 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     1501 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     1561 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     1621 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
     1681 agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     1741 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     1801 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1861 tcgtgccagc ggatcgacag tactaagctt ggtctaatac ccaatctctc gtcttatcca
     1921 gatgttttat acgccttaat taatagggat aacagggtaa tctcgaggtt taaacgtcga
     1981 cgcggccgct actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct
     2041 tcctagtctc ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg
     2101 tgacacaccc ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt
     2161 acgatggagc cagagaggat cctgggaggg agagcttggc agggggtggg agggaagggg
     2221 gggatgcgtg acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg
     2281 agctgctctg acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac
     2341 acttcccaag aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc
     2401 taactttggc tcttcacctt tctagtcccc aatttatatt gttcctccgt gcgtcagttt
     2461 tacctgtgag ataaggccag tagccagccc cgtcctggca gggctgtggt gaggaggggg
     2521 gtgtccgtgt ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt
     2581 ggccgcctct actccctttc tctttctcca tccttctttc cttaaagagt ccccagtgct
     2641 atctgggaca tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg
     2701 cttctgggtt tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt
     2761 cctcgtccac catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct
     2821 ctgctctcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg
     2881 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2941 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3001 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3061 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3121 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3181 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3241 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3301 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3361 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3421 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3481 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3541 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3601 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3661 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3721 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3781 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3841 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3901 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3961 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4021 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4081 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4141 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4201 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4261 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4321 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4381 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4441 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4501 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4561 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4621 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4681 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4741 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4801 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4861 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4921 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4981 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5041 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5101 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5161 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5221 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5281 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5341 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5401 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5461 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     5521 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     5581 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     5641 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt
     5701 acctgctttc tctgaccagc attctctccc ctgggcctgt gccgctttct gtctgcagct
     5761 tgtggcctgg gtcacctcta cggctggccc agatccttcc ctgccgcctc cttcaggttc
     5821 cgtcttcctc cactccctct tccccttgct ctctgctgtg ttgctgccca aggatgctct
     5881 ttccggagca cttccttctc ggcgctgcac cacgtgatgt cctctgagcg gatcctcccc
     5941 gtgtctgggt cctctccggg catctctcct ccctcaccca accccatgcc gtcttcactc
     6001 gctgggttcc cttttccttc tccttctggg gcctgtgcca tctctcgttt cttaggatgg
     6061 ccttctccga cggatgtctc ccttgcgtcc cgcctcccct tcttgtaggc ctgcatcatc
     6121 accgtttttc tggacaaccc caaagtaccc cgtctccctg gctttagcca cctctccatc
     6181 ctcttgcttt ctttgcctgg acaccccgtt ctcctgtgga ttcgggtcac ctctcactcc
     6241 tttcatttgg gcagctcccc tacccccctt acctctctag tctgtgctag ctcttccagc
     6301 cccctgtcat ggcatcttcc aggggtccga gagctcagct agtcttcttc ctccaacccg
     6361 ggcccctatg tccacttcag gacagcatgt ttgctgcctc cagggatcct gtgtccccga
     6421 gctgggacca ccttatattc ccagggccgg ttaatgtggc tctggttctg ggtactttta
     6481 tctgtcccct ccaccccaca gtggggcaag cttattaccc tgttatccct aaccggtgcg
     6541 tttttatgct tgtagtattg tataatgttt ttaagatcca agcttctgac ctcttctctt
     6601 cctcccacag ggcctcgaga caattgatta acatcgatac ggtaccgagt tggcgcgcct
     6661 gggagctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc
     6721 cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat
     6781 ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt
     6841 tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa
     6901 aagctttagg ctgaaagaga gatttagaat gacaggcgcg cctggccata catcgatacg
     6961 gtaccgagtt ggcgcgcctg ggagctcacg gggacagccc ccccccaaag cccccaggga
     7021 tgtaattacg tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg
     7081 tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga
     7141 aggtggcacg ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac
     7201 ctggggggat acggggaaaa agctttaggc tgaaagagag atttagaatg acaggcgcgc
     7261 ctggtggcga acgat
//
