LOCUS       40924_42069        7285 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT12_T12_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  JOURNAL   Unpublished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            "Unpublished"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    16..36
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             78..95
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             96..791
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(834..858)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1013..1068
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1429..1445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1453..1469
                     /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(1477..1507)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1522..1543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    1716..2552
                     /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(2582..2600)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2621..2637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2645..2661)
                     /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(2669..2699)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2714..2735)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3023..3611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3785..4642)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4643..4747)
                     /label=AmpR promoter
     rep_origin      complement(4773..5228)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5370..5386
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5396..5414
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5429..6232
                     /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    6311..6336
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    7001..7021
                     /label=attB4
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     protein_bind    7069..7163
                     /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    7177..7194
                     /label=TALER12 bs binding site
                     /bound_moiety="TALER12 bs"
     misc_feature    7201..7260
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    7267..7284
                     /label=TALER12 bs binding site
                     /bound_moiety="TALER12 bs"
ORIGIN
        1 gatccaattc gaccccaagt ttgtacaaaa aagcagggat cccggtaccc tcgacacctg
       61 ctgacggagc caccatgcac catcaccatc accatatggt gagcgagctg attaaggaga
      121 acatgcacat gaagctgtac atggagggca ccgtgaacaa ccaccacttc aagtgcacat
      181 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gcggtcgagg
      241 gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac ggcagcaaaa
      301 ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc cccgagggct
      361 tcacatggga gagagtcacc acatacgaag atgggggcgt gctgaccgct acccaggaca
      421 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcccat
      481 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggcctccacc gagacactgt
      541 accccgctga cggcggcctg gaaggcagag ccgacatggc cctgaagctc gtgggcgggg
      601 gccacctgat ctgcaacctt aagaccacat acagatccaa gaaacccgct aagaacctca
      661 agatgcccgg cgtctactat gtggacagga gactggaaag aatcaaggag gccgacaaag
      721 agacatacgt cgagcagcac gaggtggctg tggccagata ctgcgacctc cctagcaaac
      781 tggggcacaa acttaattcc tagtaacgct cagtgcaggt gaattctacc accacccagc
      841 tttcttgtac aaagtggtac gcgtgaattc actcctcagg tgcaggctgc ctatcagaag
      901 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
      961 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     1021 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     1081 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     1141 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
     1201 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
     1261 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
     1321 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
     1381 ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt catagctgtt
     1441 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     1501 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     1561 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc gacagtacta agcttggtga
     1621 attcccttat gtgagtgtaa aaggcaggcg agtttgtccc ttaattaata gggataacag
     1681 ggtaatctcg aggtttaaac gtcgacgcgg ccgctactag ggacaggatt ggtgacagaa
     1741 aagccccatc cttaggcctc ctccttccta gtctcctgat attgggtcta acccccacct
     1801 cctgttaggc agattcctta tctggtgaca cacccccatt tcctggagcc atctctctcc
     1861 ttgccagaac ctctaaggtt tgcttacgat ggagccagag aggatcctgg gagggagagc
     1921 ttggcagggg gtgggaggga agggggggat gcgtgacctg cccggttctc agtggccacc
     1981 ctgcgctacc ctctcccaga acctgagctg ctctgacgcg gctgtctggt gcgtttcact
     2041 gatcctggtg ctgcagcttc cttacacttc ccaagaggag aagcagtttg gaaaaacaaa
     2101 atcagaataa gttggtcctg agttctaact ttggctcttc acctttctag tccccaattt
     2161 atattgttcc tccgtgcgtc agttttacct gtgagataag gccagtagcc agccccgtcc
     2221 tggcagggct gtggtgagga ggggggtgtc cgtgtggaaa actccctttg tgagaatggt
     2281 gcgtcctagg tgttcaccag gtcgtggccg cctctactcc ctttctcttt ctccatcctt
     2341 ctttccttaa agagtcccca gtgctatctg ggacatattc ctccgcccag agcagggtcc
     2401 cgcttcccta aggccctgct ctgggcttct gggtttgagt ccttggcaag cccaggagag
     2461 gcgctcaggc ttccctgtcc cccttcctcg tccaccatct catgcccctg gctctcctgc
     2521 cccttcccta caggggttcc tggctctgct ctccaccgcg gtggagctcc agcttttgtt
     2581 ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     2641 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     2701 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     2761 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     2821 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2881 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2941 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3001 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3061 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3121 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3181 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3241 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3301 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3361 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3421 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3481 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3541 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3601 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3661 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3721 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3781 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3841 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3901 ccagtgctgc aatgataccg cgagagccac gctcaccggc tccagattta tcagcaataa
     3961 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4021 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4081 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4141 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4201 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4261 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4321 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4381 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4441 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4501 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4561 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4621 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4681 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4741 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     4801 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     4861 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     4921 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     4981 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     5041 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5101 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     5161 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5221 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     5281 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     5341 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata
     5401 cgactcacta tagggcgaat tgggtacctg ctttctctga ccagcattct ctcccctggg
     5461 cctgtgccgc tttctgtctg cagcttgtgg cctgggtcac ctctacggct ggcccagatc
     5521 cttccctgcc gcctccttca ggttccgtct tcctccactc cctcttcccc ttgctctctg
     5581 ctgtgttgct gcccaaggat gctctttccg gagcacttcc ttctcggcgc tgcaccacgt
     5641 gatgtcctct gagcggatcc tccccgtgtc tgggtcctct ccgggcatct ctcctccctc
     5701 acccaacccc atgccgtctt cactcgctgg gttccctttt ccttctcctt ctggggcctg
     5761 tgccatctct cgtttcttag gatggccttc tccgacggat gtctcccttg cgtcccgcct
     5821 ccccttcttg taggcctgca tcatcaccgt ttttctggac aaccccaaag taccccgtct
     5881 ccctggcttt agccacctct ccatcctctt gctttctttg cctggacacc ccgttctcct
     5941 gtggattcgg gtcacctctc actcctttca tttgggcagc tcccctaccc cccttacctc
     6001 tctagtctgt gctagctctt ccagccccct gtcatggcat cttccagggg tccgagagct
     6061 cagctagtct tcttcctcca acccgggccc ctatgtccac ttcaggacag catgtttgct
     6121 gcctccaggg atcctgtgtc cccgagctgg gaccacctta tattcccagg gccggttaat
     6181 gtggctctgg ttctgggtac ttttatctgt cccctccacc ccacagtggg gcaagcttat
     6241 taccctgtta tccctaaccg gtctaatacc caatctctcg tcttatccag atgttttata
     6301 cgccaagctt ctgacctctt ctcttcctcc cacagggcct cgagacaatt gattaacatc
     6361 gatacggtac cgagttggcg cgcctgggag ctcacgggga cagccccccc ccaaagcccc
     6421 cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg
     6481 ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca
     6541 cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc
     6601 agacacctgg ggggatacgg ggaaaaagct ttaggctgaa agagagattt agaatgacag
     6661 gcgcgcctgg ccatacatcg atacggtacc gagttggcgc gcctgggagc tcacggggac
     6721 agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag
     6781 cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg
     6841 tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct
     6901 cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaagctt taggctgaaa
     6961 gagagattta gaatgacagg cgcgcctggt ggcgaacgat caactttgta tagaaaagtt
     7021 ggctccgaat ttctcgacag atctcatgtg attacgccaa gctacgggcg gagtactgtc
     7081 ctccgagcgg agtactgtcc tccgagcgga gtactgtcct ccgagcggag tactgtcctc
     7141 cgagcggagt tctgtcctcc gagcggagac tctagatatt tcgacgctca ttctgaattc
     7201 taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc gtcagatcgc
     7261 ctcgagtatt tcgacgctca ttctg
//
