LOCUS       40924_42244        7310 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT2_T2x3_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7310)
  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  JOURNAL   Unpublished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 7310)
  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 7310)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7310)
  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            "Unpublished"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..7310
                     /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..190
                     /label=TALER2 bs binding site
                     /bound_moiety="TALER2 bs"
     misc_feature    197..256
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    263..276
                     /label=TALER2 bs binding site
                     /bound_moiety="TALER2 bs"
     protein_bind    279..292
                     /label=TALER2 bs binding site
                     /bound_moiety="TALER2 bs"
     protein_bind    295..308
                     /label=TALER2 bs binding site
                     /bound_moiety="TALER2 bs"
     protein_bind    326..346
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             388..405
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             406..1101
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1144..1168)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1323..1378
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1739..1755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1763..1779
                     /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(1787..1817)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1832..1853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2026..2862
                     /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(2892..2910)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2931..2947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2955..2971)
                     /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(2979..3009)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3024..3045)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3333..3921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4095..4952)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4953..5057)
                     /label=AmpR promoter
     rep_origin      complement(5083..5538)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5680..5696
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5706..5724
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5739..6542
                     /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    6621..6646
                     /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 tctagataag
      181 aataaatata gaattctagg cgtgtacggt gggaggccta tataagcaga gctcgtttag
      241 tgaaccgtca gatcgcctcg agtaagaata aatataccta agaataaata tacgtaagaa
      301 taaatatagg gatccaattc gaccccaagt ttgtacaaaa aagcagggat cccggtaccc
      361 tcgacacctg ctgacggagc caccatgcac catcaccatc accatatggt gagcgagctg
      421 attaaggaga acatgcacat gaagctgtac atggagggca ccgtgaacaa ccaccacttc
      481 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
      541 gcggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac
      601 ggcagcaaaa ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc
      661 cccgagggct tcacatggga gagagtcacc acatacgaag atgggggcgt gctgaccgct
      721 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
      781 aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggcctccacc
      841 gagacactgt accccgctga cggcggcctg gaaggcagag ccgacatggc cctgaagctc
      901 gtgggcgggg gccacctgat ctgcaacctt aagaccacat acagatccaa gaaacccgct
      961 aagaacctca agatgcccgg cgtctactat gtggacagga gactggaaag aatcaaggag
     1021 gccgacaaag agacatacgt cgagcagcac gaggtggctg tggccagata ctgcgacctc
     1081 cctagcaaac tggggcacaa acttaattcc tagtaacgct cagtgcaggt gaattctacc
     1141 accacccagc tttcttgtac aaagtggtac gcgtgaattc actcctcagg tgcaggctgc
     1201 ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct
     1261 ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg
     1321 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact
     1381 cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag
     1441 tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca
     1501 tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg
     1561 aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt
     1621 ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc
     1681 tgtccctctt ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt
     1741 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     1801 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1861 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc gacagtacta
     1921 agcttggtct aatacccaat ctctcgtctt atccagatgt tttatacgcc ttaattaata
     1981 gggataacag ggtaatctcg aggtttaaac gtcgacgcgg ccgctactag ggacaggatt
     2041 ggtgacagaa aagccccatc cttaggcctc ctccttccta gtctcctgat attgggtcta
     2101 acccccacct cctgttaggc agattcctta tctggtgaca cacccccatt tcctggagcc
     2161 atctctctcc ttgccagaac ctctaaggtt tgcttacgat ggagccagag aggatcctgg
     2221 gagggagagc ttggcagggg gtgggaggga agggggggat gcgtgacctg cccggttctc
     2281 agtggccacc ctgcgctacc ctctcccaga acctgagctg ctctgacgcg gctgtctggt
     2341 gcgtttcact gatcctggtg ctgcagcttc cttacacttc ccaagaggag aagcagtttg
     2401 gaaaaacaaa atcagaataa gttggtcctg agttctaact ttggctcttc acctttctag
     2461 tccccaattt atattgttcc tccgtgcgtc agttttacct gtgagataag gccagtagcc
     2521 agccccgtcc tggcagggct gtggtgagga ggggggtgtc cgtgtggaaa actccctttg
     2581 tgagaatggt gcgtcctagg tgttcaccag gtcgtggccg cctctactcc ctttctcttt
     2641 ctccatcctt ctttccttaa agagtcccca gtgctatctg ggacatattc ctccgcccag
     2701 agcagggtcc cgcttcccta aggccctgct ctgggcttct gggtttgagt ccttggcaag
     2761 cccaggagag gcgctcaggc ttccctgtcc cccttcctcg tccaccatct catgcccctg
     2821 gctctcctgc cccttcccta caggggttcc tggctctgct ctccaccgcg gtggagctcc
     2881 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
     2941 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3001 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3061 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3121 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3181 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3241 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3301 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3361 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3421 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3481 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3541 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3601 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3661 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3721 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3781 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3841 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3901 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3961 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4021 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4081 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4141 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4201 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4261 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4321 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4381 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4441 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4501 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4561 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4621 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4681 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4741 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4801 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4861 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4921 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4981 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5041 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     5101 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5161 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5221 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5281 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5341 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5401 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5461 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5521 cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     5581 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     5641 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc
     5701 gcgcgtaata cgactcacta tagggcgaat tgggtacctg ctttctctga ccagcattct
     5761 ctcccctggg cctgtgccgc tttctgtctg cagcttgtgg cctgggtcac ctctacggct
     5821 ggcccagatc cttccctgcc gcctccttca ggttccgtct tcctccactc cctcttcccc
     5881 ttgctctctg ctgtgttgct gcccaaggat gctctttccg gagcacttcc ttctcggcgc
     5941 tgcaccacgt gatgtcctct gagcggatcc tccccgtgtc tgggtcctct ccgggcatct
     6001 ctcctccctc acccaacccc atgccgtctt cactcgctgg gttccctttt ccttctcctt
     6061 ctggggcctg tgccatctct cgtttcttag gatggccttc tccgacggat gtctcccttg
     6121 cgtcccgcct ccccttcttg taggcctgca tcatcaccgt ttttctggac aaccccaaag
     6181 taccccgtct ccctggcttt agccacctct ccatcctctt gctttctttg cctggacacc
     6241 ccgttctcct gtggattcgg gtcacctctc actcctttca tttgggcagc tcccctaccc
     6301 cccttacctc tctagtctgt gctagctctt ccagccccct gtcatggcat cttccagggg
     6361 tccgagagct cagctagtct tcttcctcca acccgggccc ctatgtccac ttcaggacag
     6421 catgtttgct gcctccaggg atcctgtgtc cccgagctgg gaccacctta tattcccagg
     6481 gccggttaat gtggctctgg ttctgggtac ttttatctgt cccctccacc ccacagtggg
     6541 gcaagcttat taccctgtta tccctaaccg gtgcgttttt atgcttgtag tattgtataa
     6601 tgtttttaag atccaagctt ctgacctctt ctcttcctcc cacagggcct cgagacaatt
     6661 gattaacatc gatacggtac cgagttggcg cgcctgggag ctcacgggga cagccccccc
     6721 ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc
     6781 cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac
     6841 agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct
     6901 ttgagcctgc agacacctgg ggggatacgg ggaaaaagct ttaggctgaa agagagattt
     6961 agaatgacag gcgcgcctgg ccatacatcg atacggtacc gagttggcgc gcctgggagc
     7021 tcacggggac agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag
     7081 ggggcagcag cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga
     7141 gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct
     7201 gaacgcttct cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaagctt
     7261 taggctgaaa gagagattta gaatgacagg cgcgcctggt ggcgaacgat
//
