LOCUS       40924_42329        7285 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT5_T5_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7285)
  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  JOURNAL   Unpublished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 7285)
  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 7285)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7285)
  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            "Unpublished"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..7285
                     /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    301..321
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             363..380
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             381..1076
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1119..1143)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1298..1353
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1714..1730)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1738..1754
                     /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(1762..1792)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1807..1828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2001..2837
                     /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(2867..2885)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2906..2922)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2930..2946)
                     /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(2954..2984)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2999..3020)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3308..3896)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4070..4927)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4928..5032)
                     /label=AmpR promoter
     rep_origin      complement(5058..5513)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5655..5671
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5681..5699
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5714..6517
                     /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    6596..6621
                     /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 cactggatcc aattcgaccc
      301 caagtttgta caaaaaagca gggatcccgg taccctcgac acctgctgac ggagccacca
      361 tgcaccatca ccatcaccat atggtgagcg agctgattaa ggagaacatg cacatgaagc
      421 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
      481 agccctacga gggcacccag accatgagaa tcaaggcggt cgagggcggc cctctcccct
      541 tcgccttcga catcctggct accagcttca tgtacggcag caaaaccttc atcaaccaca
      601 cccagggcat ccccgacttc tttaagcagt ccttccccga gggcttcaca tgggagagag
      661 tcaccacata cgaagatggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
      721 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
      781 tgcagaagaa aacactcggc tgggaggcct ccaccgagac actgtacccc gctgacggcg
      841 gcctggaagg cagagccgac atggccctga agctcgtggg cgggggccac ctgatctgca
      901 accttaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
      961 actatgtgga caggagactg gaaagaatca aggaggccga caaagagaca tacgtcgagc
     1021 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta
     1081 attcctagta acgctcagtg caggtgaatt ctaccaccac ccagctttct tgtacaaagt
     1141 ggtacgcgtg aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg
     1201 gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg
     1261 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     1321 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     1381 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg
     1441 gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct
     1501 gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt
     1561 tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga
     1621 tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc
     1681 ctcgacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     1741 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     1801 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1861 gggaaacctg tcgtgccagc ggatcgacag tactaagctt ggtctaatac ccaatctctc
     1921 gtcttatcca gatgttttat acgccttaat taatagggat aacagggtaa tctcgaggtt
     1981 taaacgtcga cgcggccgct actagggaca ggattggtga cagaaaagcc ccatccttag
     2041 gcctcctcct tcctagtctc ctgatattgg gtctaacccc cacctcctgt taggcagatt
     2101 ccttatctgg tgacacaccc ccatttcctg gagccatctc tctccttgcc agaacctcta
     2161 aggtttgctt acgatggagc cagagaggat cctgggaggg agagcttggc agggggtggg
     2221 agggaagggg gggatgcgtg acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc
     2281 ccagaacctg agctgctctg acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca
     2341 gcttccttac acttcccaag aggagaagca gtttggaaaa acaaaatcag aataagttgg
     2401 tcctgagttc taactttggc tcttcacctt tctagtcccc aatttatatt gttcctccgt
     2461 gcgtcagttt tacctgtgag ataaggccag tagccagccc cgtcctggca gggctgtggt
     2521 gaggaggggg gtgtccgtgt ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc
     2581 accaggtcgt ggccgcctct actccctttc tctttctcca tccttctttc cttaaagagt
     2641 ccccagtgct atctgggaca tattcctccg cccagagcag ggtcccgctt ccctaaggcc
     2701 ctgctctggg cttctgggtt tgagtccttg gcaagcccag gagaggcgct caggcttccc
     2761 tgtccccctt cctcgtccac catctcatgc ccctggctct cctgcccctt ccctacaggg
     2821 gttcctggct ctgctctcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt
     2881 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     2941 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3001 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3061 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3121 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3181 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3241 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3301 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3361 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3421 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3481 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3541 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3601 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3661 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3721 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3781 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3841 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3901 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3961 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4021 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4081 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4141 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4201 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4261 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4321 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4381 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4441 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4501 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4561 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4621 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4681 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4741 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4801 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4861 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4921 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4981 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5041 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5101 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5161 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5221 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5281 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5341 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5401 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5461 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     5521 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     5581 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     5641 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg
     5701 cgaattgggt acctgctttc tctgaccagc attctctccc ctgggcctgt gccgctttct
     5761 gtctgcagct tgtggcctgg gtcacctcta cggctggccc agatccttcc ctgccgcctc
     5821 cttcaggttc cgtcttcctc cactccctct tccccttgct ctctgctgtg ttgctgccca
     5881 aggatgctct ttccggagca cttccttctc ggcgctgcac cacgtgatgt cctctgagcg
     5941 gatcctcccc gtgtctgggt cctctccggg catctctcct ccctcaccca accccatgcc
     6001 gtcttcactc gctgggttcc cttttccttc tccttctggg gcctgtgcca tctctcgttt
     6061 cttaggatgg ccttctccga cggatgtctc ccttgcgtcc cgcctcccct tcttgtaggc
     6121 ctgcatcatc accgtttttc tggacaaccc caaagtaccc cgtctccctg gctttagcca
     6181 cctctccatc ctcttgcttt ctttgcctgg acaccccgtt ctcctgtgga ttcgggtcac
     6241 ctctcactcc tttcatttgg gcagctcccc tacccccctt acctctctag tctgtgctag
     6301 ctcttccagc cccctgtcat ggcatcttcc aggggtccga gagctcagct agtcttcttc
     6361 ctccaacccg ggcccctatg tccacttcag gacagcatgt ttgctgcctc cagggatcct
     6421 gtgtccccga gctgggacca ccttatattc ccagggccgg ttaatgtggc tctggttctg
     6481 ggtactttta tctgtcccct ccaccccaca gtggggcaag cttattaccc tgttatccct
     6541 aaccggtgcg tttttatgct tgtagtattg tataatgttt ttaagatcca agcttctgac
     6601 ctcttctctt cctcccacag ggcctcgaga caattgatta acatcgatac ggtaccgagt
     6661 tggcgcgcct gggagctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac
     6721 gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct
     6781 ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac
     6841 gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggga
     6901 tacggggaaa aagctttagg ctgaaagaga gatttagaat gacaggcgcg cctggccata
     6961 catcgatacg gtaccgagtt ggcgcgcctg ggagctcacg gggacagccc ccccccaaag
     7021 cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc cgcccggggc
     7081 tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg
     7141 ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg ctctttgagc
     7201 ctgcagacac ctggggggat acggggaaaa agctttaggc tgaaagagag atttagaatg
     7261 acaggcgcgc ctggtggcga acgat
//
