LOCUS       40924_42154        7283 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT1_T1_78bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7283)
  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  JOURNAL   Unpublished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 7283)
  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 7283)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7283)
  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            "Unpublished"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..7283
                     /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    231..251
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             293..310
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             311..1006
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1049..1073)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1228..1283
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1644..1660)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1668..1684
                     /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(1692..1722)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1737..1758
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    1931..2767
                     /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(2797..2815)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2836..2852)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2860..2876)
                     /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(2884..2914)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2929..2950)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3238..3826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4000..4857)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4858..4962)
                     /label=AmpR promoter
     rep_origin      complement(4988..5443)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5585..5601
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5611..5629
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5644..6447
                     /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    6526..6551
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    7216..7236
                     /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 tacgactcac tataggatcc aattcgaccc caagtttgta
      241 caaaaaagca gggatcccgg taccctcgac acctgctgac ggagccacca tgcaccatca
      301 ccatcaccat atggtgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
      361 gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga
      421 gggcacccag accatgagaa tcaaggcggt cgagggcggc cctctcccct tcgccttcga
      481 catcctggct accagcttca tgtacggcag caaaaccttc atcaaccaca cccagggcat
      541 ccccgacttc tttaagcagt ccttccccga gggcttcaca tgggagagag tcaccacata
      601 cgaagatggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
      661 ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa
      721 aacactcggc tgggaggcct ccaccgagac actgtacccc gctgacggcg gcctggaagg
      781 cagagccgac atggccctga agctcgtggg cgggggccac ctgatctgca accttaagac
      841 cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga
      901 caggagactg gaaagaatca aggaggccga caaagagaca tacgtcgagc agcacgaggt
      961 ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta attcctagta
     1021 acgctcagtg caggtgaatt ctaccaccac ccagctttct tgtacaaagt ggtacgcgtg
     1081 aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     1141 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     1201 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     1261 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     1321 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     1381 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     1441 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     1501 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     1561 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
     1621 agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     1681 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     1741 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1801 tcgtgccagc ggatcgacag tactaagctt ggtgaattcc cttatgtgag tgtaaaaggc
     1861 aggcgagttt gtcccttaat taatagggat aacagggtaa tctcgaggtt taaacgtcga
     1921 cgcggccgct actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct
     1981 tcctagtctc ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg
     2041 tgacacaccc ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt
     2101 acgatggagc cagagaggat cctgggaggg agagcttggc agggggtggg agggaagggg
     2161 gggatgcgtg acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg
     2221 agctgctctg acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac
     2281 acttcccaag aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc
     2341 taactttggc tcttcacctt tctagtcccc aatttatatt gttcctccgt gcgtcagttt
     2401 tacctgtgag ataaggccag tagccagccc cgtcctggca gggctgtggt gaggaggggg
     2461 gtgtccgtgt ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt
     2521 ggccgcctct actccctttc tctttctcca tccttctttc cttaaagagt ccccagtgct
     2581 atctgggaca tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg
     2641 cttctgggtt tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt
     2701 cctcgtccac catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct
     2761 ctgctctcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg
     2821 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2881 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2941 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3001 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3061 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3121 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3181 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3241 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3301 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3361 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3421 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3481 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3541 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3601 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3661 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3721 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3781 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3841 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3901 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3961 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4021 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4081 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4141 gccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4201 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4261 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4321 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4381 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4441 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4501 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4561 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4621 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4681 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4741 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4801 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4861 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4921 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4981 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5041 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5101 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5161 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5221 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5281 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5341 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5401 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     5461 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     5521 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     5581 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt
     5641 acctgctttc tctgaccagc attctctccc ctgggcctgt gccgctttct gtctgcagct
     5701 tgtggcctgg gtcacctcta cggctggccc agatccttcc ctgccgcctc cttcaggttc
     5761 cgtcttcctc cactccctct tccccttgct ctctgctgtg ttgctgccca aggatgctct
     5821 ttccggagca cttccttctc ggcgctgcac cacgtgatgt cctctgagcg gatcctcccc
     5881 gtgtctgggt cctctccggg catctctcct ccctcaccca accccatgcc gtcttcactc
     5941 gctgggttcc cttttccttc tccttctggg gcctgtgcca tctctcgttt cttaggatgg
     6001 ccttctccga cggatgtctc ccttgcgtcc cgcctcccct tcttgtaggc ctgcatcatc
     6061 accgtttttc tggacaaccc caaagtaccc cgtctccctg gctttagcca cctctccatc
     6121 ctcttgcttt ctttgcctgg acaccccgtt ctcctgtgga ttcgggtcac ctctcactcc
     6181 tttcatttgg gcagctcccc tacccccctt acctctctag tctgtgctag ctcttccagc
     6241 cccctgtcat ggcatcttcc aggggtccga gagctcagct agtcttcttc ctccaacccg
     6301 ggcccctatg tccacttcag gacagcatgt ttgctgcctc cagggatcct gtgtccccga
     6361 gctgggacca ccttatattc ccagggccgg ttaatgtggc tctggttctg ggtactttta
     6421 tctgtcccct ccaccccaca gtggggcaag cttattaccc tgttatccct aaccggtcta
     6481 atacccaatc tctcgtctta tccagatgtt ttatacgcca agcttctgac ctcttctctt
     6541 cctcccacag ggcctcgaga caattgatta acatcgatac ggtaccgagt tggcgcgcct
     6601 gggagctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc
     6661 cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat
     6721 ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt
     6781 tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa
     6841 aagctttagg ctgaaagaga gatttagaat gacaggcgcg cctggccata catcgatacg
     6901 gtaccgagtt ggcgcgcctg ggagctcacg gggacagccc ccccccaaag cccccaggga
     6961 tgtaattacg tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg
     7021 tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga
     7081 aggtggcacg ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac
     7141 ctggggggat acggggaaaa agctttaggc tgaaagagag atttagaatg acaggcgcgc
     7201 ctggtggcga acgatcaact ttgtatagaa aagttggctc cgaatttctc gacagatctc
     7261 atgtgattac gccaagctac ggg
//
