LOCUS       40924_42064        7339 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT12_T12x3_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7339)
  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  JOURNAL   Unpublished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 7339)
  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 7339)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7339)
  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            "Unpublished"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..7339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(222..810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(984..1841)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1842..1946)
                     /label=AmpR promoter
     rep_origin      complement(1972..2427)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     2569..2585
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2595..2613
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2628..3431
                     /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    3510..3535
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    4207..4227
                     /label=attB4
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     protein_bind    4275..4369
                     /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    4383..4400
                     /label=TALER12 bs binding site
                     /bound_moiety="TALER12 bs"
     misc_feature    4407..4466
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    4473..4490
                     /label=TALER12 bs binding site
                     /bound_moiety="TALER12 bs"
     protein_bind    4491..4508
                     /label=TALER12 bs binding site
                     /bound_moiety="TALER12 bs"
     protein_bind    4509..4526
                     /label=TALER12 bs binding site
                     /bound_moiety="TALER12 bs"
     protein_bind    4541..4565
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4583..5278
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     misc_feature    5293..5349
                     /label=MCS
                     /note="multiple cloning site"
     protein_bind    complement(5372..5396)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    5551..5606
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5967..5983)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5991..6007
                     /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(6015..6045)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6060..6081
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    6254..7090
                     /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(7120..7138)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7159..7175)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7183..7199)
                     /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(7207..7237)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7252..7273)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
ORIGIN
        1 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
       61 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      121 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      181 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      241 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      301 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      361 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      421 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      481 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      541 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      601 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      661 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
      721 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
      781 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
      841 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
      901 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
      961 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1021 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1081 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     1141 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1201 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1261 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1321 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1381 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1441 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1501 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1561 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1621 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1681 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     1741 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     1801 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     1861 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     1921 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     1981 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2041 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2101 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2161 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2221 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2281 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2341 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2401 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     2461 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     2521 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     2581 ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtacctgc tttctctgac
     2641 cagcattctc tcccctgggc ctgtgccgct ttctgtctgc agcttgtggc ctgggtcacc
     2701 tctacggctg gcccagatcc ttccctgccg cctccttcag gttccgtctt cctccactcc
     2761 ctcttcccct tgctctctgc tgtgttgctg cccaaggatg ctctttccgg agcacttcct
     2821 tctcggcgct gcaccacgtg atgtcctctg agcggatcct ccccgtgtct gggtcctctc
     2881 cgggcatctc tcctccctca cccaacccca tgccgtcttc actcgctggg ttcccttttc
     2941 cttctccttc tggggcctgt gccatctctc gtttcttagg atggccttct ccgacggatg
     3001 tctcccttgc gtcccgcctc cccttcttgt aggcctgcat catcaccgtt tttctggaca
     3061 accccaaagt accccgtctc cctggcttta gccacctctc catcctcttg ctttctttgc
     3121 ctggacaccc cgttctcctg tggattcggg tcacctctca ctcctttcat ttgggcagct
     3181 cccctacccc ccttacctct ctagtctgtg ctagctcttc cagccccctg tcatggcatc
     3241 ttccaggggt ccgagagctc agctagtctt cttcctccaa cccgggcccc tatgtccact
     3301 tcaggacagc atgtttgctg cctccaggga tcctgtgtcc ccgagctggg accaccttat
     3361 attcccaggg ccggttaatg tggctctggt tctgggtact tttatctgtc ccctccaccc
     3421 cacagtgggg caagcttatt accctgttat ccctaaccgg tgcgttttta tgcttgtagt
     3481 attgtataat gtttttaaga tccaagcttc tgacctcttc tcttcctccc acagggcctc
     3541 gagacaattg catcgatggt accagtgaaa gtctgcaatg aattcgagtt ggcgcgcctg
     3601 tcattctaaa tctctctttc agcctaaagc tttttccccg tatcccccca ggtgtctgca
     3661 ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc
     3721 ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg
     3781 agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg
     3841 gctttgggag ggggctgtcc ccgtgagctc ccaggcgcgc ctgtcattct aaatctctct
     3901 ttcagcctaa agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga
     3961 gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac
     4021 gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc
     4081 tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg
     4141 tccccgtgag ctcccaggcg cgccaactcg gtaccgtatc gatgtcgacg ttaacgctag
     4201 tgatatcaac tttgtataga aaagttggct ccgaatttct cgacagatct catgtgatta
     4261 cgccaagcta cgggcggagt actgtcctcc gagcggagta ctgtcctccg agcggagtac
     4321 tgtcctccga gcggagtact gtcctccgag cggagttctg tcctccgagc ggagactcta
     4381 gatatttcga cgctcattct gaattctagg cgtgtacggt gggaggccta tataagcaga
     4441 gctcgtttag tgaaccgtca gatcgcctcg agtatttcga cgctcattct tatttcgacg
     4501 ctcattctta tttcgacgct cattctggat ccaattcgac ccaagtttgt acaaaaaagc
     4561 aggctgatta ccggtcgcca ccatggtgag cgagctgatt aaggagaaca tgcacatgaa
     4621 gctgtacatg gagggcaccg tgaacaacca ccacttcaag tgcacatccg agggcgaagg
     4681 caagccctac gagggcaccc agaccatgag aatcaaggcg gtcgagggcg gccctctccc
     4741 cttcgccttc gacatcctgg ctaccagctt catgtacggc agcaaaacct tcatcaacca
     4801 cacccagggc atccccgact tctttaagca gtccttcccc gagggcttca catgggagag
     4861 agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga
     4921 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcccatcca acggccctgt
     4981 gatgcagaag aaaacactcg gctgggaggc ctccaccgag accctgtacc ccgctgacgg
     5041 cggcctggaa ggcagagccg acatggccct gaagctcgtg ggcgggggcc acctgatctg
     5101 caacttgaag accacataca gatccaagaa acccgctaag aacctcaaga tgcccggcgt
     5161 ctactatgtg gacagaagac tggaaagaat caaggaggcc gacaaagaga cctacgtcga
     5221 gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg ggcacagatg
     5281 agcggccgcc tcagatctcg agctcaagct tcgaattctg cagtcgacgg taccgcgggc
     5341 ccgggatcca ccggatctag ataactgatc tacccagctt tcttgtacaa agtggtacgc
     5401 gtgaattcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg
     5461 ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca
     5521 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag
     5581 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa
     5641 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca
     5701 tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt
     5761 ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt
     5821 tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc
     5881 tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc
     5941 tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     6001 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     6061 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6121 ctgtcgtgcc agcggatcga cagtactaag cttggtctaa tacccaatct ctcgtcttat
     6181 ccagatgttt tatacgcctt aattaatagg gataacaggg taatctcgag gtttaaacgt
     6241 cgacgcggcc gctactaggg acaggattgg tgacagaaaa gccccatcct taggcctcct
     6301 ccttcctagt ctcctgatat tgggtctaac ccccacctcc tgttaggcag attccttatc
     6361 tggtgacaca cccccatttc ctggagccat ctctctcctt gccagaacct ctaaggtttg
     6421 cttacgatgg agccagagag gatcctggga gggagagctt ggcagggggt gggagggaag
     6481 ggggggatgc gtgacctgcc cggttctcag tggccaccct gcgctaccct ctcccagaac
     6541 ctgagctgct ctgacgcggc tgtctggtgc gtttcactga tcctggtgct gcagcttcct
     6601 tacacttccc aagaggagaa gcagtttgga aaaacaaaat cagaataagt tggtcctgag
     6661 ttctaacttt ggctcttcac ctttctagtc cccaatttat attgttcctc cgtgcgtcag
     6721 ttttacctgt gagataaggc cagtagccag ccccgtcctg gcagggctgt ggtgaggagg
     6781 ggggtgtccg tgtggaaaac tccctttgtg agaatggtgc gtcctaggtg ttcaccaggt
     6841 cgtggccgcc tctactccct ttctctttct ccatccttct ttccttaaag agtccccagt
     6901 gctatctggg acatattcct ccgcccagag cagggtcccg cttccctaag gccctgctct
     6961 gggcttctgg gtttgagtcc ttggcaagcc caggagaggc gctcaggctt ccctgtcccc
     7021 cttcctcgtc caccatctca tgcccctggc tctcctgccc cttccctaca ggggttcctg
     7081 gctctgctct ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc
     7141 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     7201 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     7261 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     7321 ccagctgcat taatgaatc
//
