LOCUS       40924_42164        7294 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT1_T1_89bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7294)
  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  JOURNAL   Unpublished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 7294)
  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 7294)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7294)
  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            "Unpublished"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..7294
                     /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    242..262
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             304..321
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             322..1017
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1060..1084)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1239..1294
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1655..1671)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1679..1695
                     /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(1703..1733)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1748..1769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    1942..2778
                     /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(2808..2826)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2847..2863)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2871..2887)
                     /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(2895..2925)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2940..2961)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3249..3837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4011..4868)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4869..4973)
                     /label=AmpR promoter
     rep_origin      complement(4999..5454)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5596..5612
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5622..5640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5655..6458
                     /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    6537..6562
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    7227..7247
                     /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 gtgtcgcgag ttacgactca ctataggatc caattcgacc
      241 ccaagtttgt acaaaaaagc agggatcccg gtaccctcga cacctgctga cggagccacc
      301 atgcaccatc accatcacca tatggtgagc gagctgatta aggagaacat gcacatgaag
      361 ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga gggcgaaggc
      421 aagccctacg agggcaccca gaccatgaga atcaaggcgg tcgagggcgg ccctctcccc
      481 ttcgccttcg acatcctggc taccagcttc atgtacggca gcaaaacctt catcaaccac
      541 acccagggca tccccgactt ctttaagcag tccttccccg agggcttcac atgggagaga
      601 gtcaccacat acgaagatgg gggcgtgctg accgctaccc aggacaccag cctccaggac
      661 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa cggccctgtg
      721 atgcagaaga aaacactcgg ctgggaggcc tccaccgaga cactgtaccc cgctgacggc
      781 ggcctggaag gcagagccga catggccctg aagctcgtgg gcgggggcca cctgatctgc
      841 aaccttaaga ccacatacag atccaagaaa cccgctaaga acctcaagat gcccggcgtc
      901 tactatgtgg acaggagact ggaaagaatc aaggaggccg acaaagagac atacgtcgag
      961 cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg gcacaaactt
     1021 aattcctagt aacgctcagt gcaggtgaat tctaccacca cccagctttc ttgtacaaag
     1081 tggtacgcgt gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt
     1141 ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg
     1201 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     1261 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     1321 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct
     1381 ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc
     1441 tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg
     1501 ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag
     1561 atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc
     1621 cctcgacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1681 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1741 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1801 cgggaaacct gtcgtgccag cggatcgaca gtactaagct tggtgaattc ccttatgtga
     1861 gtgtaaaagg caggcgagtt tgtcccttaa ttaataggga taacagggta atctcgaggt
     1921 ttaaacgtcg acgcggccgc tactagggac aggattggtg acagaaaagc cccatcctta
     1981 ggcctcctcc ttcctagtct cctgatattg ggtctaaccc ccacctcctg ttaggcagat
     2041 tccttatctg gtgacacacc cccatttcct ggagccatct ctctccttgc cagaacctct
     2101 aaggtttgct tacgatggag ccagagagga tcctgggagg gagagcttgg cagggggtgg
     2161 gagggaaggg ggggatgcgt gacctgcccg gttctcagtg gccaccctgc gctaccctct
     2221 cccagaacct gagctgctct gacgcggctg tctggtgcgt ttcactgatc ctggtgctgc
     2281 agcttcctta cacttcccaa gaggagaagc agtttggaaa aacaaaatca gaataagttg
     2341 gtcctgagtt ctaactttgg ctcttcacct ttctagtccc caatttatat tgttcctccg
     2401 tgcgtcagtt ttacctgtga gataaggcca gtagccagcc ccgtcctggc agggctgtgg
     2461 tgaggagggg ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt cctaggtgtt
     2521 caccaggtcg tggccgcctc tactcccttt ctctttctcc atccttcttt ccttaaagag
     2581 tccccagtgc tatctgggac atattcctcc gcccagagca gggtcccgct tccctaaggc
     2641 cctgctctgg gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc tcaggcttcc
     2701 ctgtccccct tcctcgtcca ccatctcatg cccctggctc tcctgcccct tccctacagg
     2761 ggttcctggc tctgctctcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     2821 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2881 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2941 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3001 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3061 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3121 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3181 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3241 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3301 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3361 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3421 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3481 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3541 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3601 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3661 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3721 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3781 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3841 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3901 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3961 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4021 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4081 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4141 ataccgcgag agccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4201 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4261 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4321 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4381 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4441 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4501 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4561 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4621 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4681 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4741 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4801 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4861 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4921 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4981 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5041 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5101 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     5161 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     5221 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     5281 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     5341 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     5401 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     5461 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     5521 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     5581 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg
     5641 gcgaattggg tacctgcttt ctctgaccag cattctctcc cctgggcctg tgccgctttc
     5701 tgtctgcagc ttgtggcctg ggtcacctct acggctggcc cagatccttc cctgccgcct
     5761 ccttcaggtt ccgtcttcct ccactccctc ttccccttgc tctctgctgt gttgctgccc
     5821 aaggatgctc tttccggagc acttccttct cggcgctgca ccacgtgatg tcctctgagc
     5881 ggatcctccc cgtgtctggg tcctctccgg gcatctctcc tccctcaccc aaccccatgc
     5941 cgtcttcact cgctgggttc ccttttcctt ctccttctgg ggcctgtgcc atctctcgtt
     6001 tcttaggatg gccttctccg acggatgtct cccttgcgtc ccgcctcccc ttcttgtagg
     6061 cctgcatcat caccgttttt ctggacaacc ccaaagtacc ccgtctccct ggctttagcc
     6121 acctctccat cctcttgctt tctttgcctg gacaccccgt tctcctgtgg attcgggtca
     6181 cctctcactc ctttcatttg ggcagctccc ctacccccct tacctctcta gtctgtgcta
     6241 gctcttccag ccccctgtca tggcatcttc caggggtccg agagctcagc tagtcttctt
     6301 cctccaaccc gggcccctat gtccacttca ggacagcatg tttgctgcct ccagggatcc
     6361 tgtgtccccg agctgggacc accttatatt cccagggccg gttaatgtgg ctctggttct
     6421 gggtactttt atctgtcccc tccaccccac agtggggcaa gcttattacc ctgttatccc
     6481 taaccggtct aatacccaat ctctcgtctt atccagatgt tttatacgcc aagcttctga
     6541 cctcttctct tcctcccaca gggcctcgag acaattgatt aacatcgata cggtaccgag
     6601 ttggcgcgcc tgggagctca cggggacagc ccccccccaa agcccccagg gatgtaatta
     6661 cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc ggtccggcgc
     6721 tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca
     6781 cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac acctgggggg
     6841 atacggggaa aaagctttag gctgaaagag agatttagaa tgacaggcgc gcctggccat
     6901 acatcgatac ggtaccgagt tggcgcgcct gggagctcac ggggacagcc cccccccaaa
     6961 gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg
     7021 ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc
     7081 gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag
     7141 cctgcagaca cctgggggga tacggggaaa aagctttagg ctgaaagaga gatttagaat
     7201 gacaggcgcg cctggtggcg aacgatcaac tttgtataga aaagttggct ccgaatttct
     7261 cgacagatct catgtgatta cgccaagcta cggg
//
