LOCUS       40924_42284        7305 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT32_T32x3_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7305)
  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  JOURNAL   Unpublished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 7305)
  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 7305)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7305)
  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            "Unpublished"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..7305
                     /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=TALER32 bs binding site
                     /bound_moiety="TALER32 bs"
     misc_feature    197..256
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    263..276
                     /label=TALER32 bs binding site
                     /bound_moiety="TALER32 bs"
     protein_bind    277..290
                     /label=TALER32 bs binding site
                     /bound_moiety="TALER32 bs"
     protein_bind    291..304
                     /label=TALER32 bs binding site
                     /bound_moiety="TALER32 bs"
     protein_bind    321..341
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             383..400
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             401..1096
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1139..1163)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1318..1373
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1734..1750)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1758..1774
                     /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(1782..1812)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1827..1848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2021..2857
                     /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(2887..2905)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2926..2942)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2950..2966)
                     /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(2974..3004)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3019..3040)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3328..3916)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4090..4947)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4948..5052)
                     /label=AmpR promoter
     rep_origin      complement(5078..5533)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5675..5691
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5701..5719
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5734..6537
                     /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    6616..6641
                     /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 tctagataca
      181 acacactata gaattctagg cgtgtacggt gggaggccta tataagcaga gctcgtttag
      241 tgaaccgtca gatcgcctcg agtacaacac actatataca acacactata tacaacacac
      301 tataggatcc aattcgaccc caagtttgta caaaaaagca gggatcccgg taccctcgac
      361 acctgctgac ggagccacca tgcaccatca ccatcaccat atggtgagcg agctgattaa
      421 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg
      481 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggcggt
      541 cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag
      601 caaaaccttc atcaaccaca cccagggcat ccccgacttc tttaagcagt ccttccccga
      661 gggcttcaca tgggagagag tcaccacata cgaagatggg ggcgtgctga ccgctaccca
      721 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
      781 cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct ccaccgagac
      841 actgtacccc gctgacggcg gcctggaagg cagagccgac atggccctga agctcgtggg
      901 cgggggccac ctgatctgca accttaagac cacatacaga tccaagaaac ccgctaagaa
      961 cctcaagatg cccggcgtct actatgtgga caggagactg gaaagaatca aggaggccga
     1021 caaagagaca tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag
     1081 caaactgggg cacaaactta attcctagta acgctcagtg caggtgaatt ctaccaccac
     1141 ccagctttct tgtacaaagt ggtacgcgtg aattcactcc tcaggtgcag gctgcctatc
     1201 agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc
     1261 cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact tctggctaat
     1321 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     1381 ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg
     1441 caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt
     1501 atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt
     1561 agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct
     1621 tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc
     1681 ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc atggtcatag
     1741 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1801 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1861 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatcgacag tactaagctt
     1921 ggtctaatac ccaatctctc gtcttatcca gatgttttat acgccttaat taatagggat
     1981 aacagggtaa tctcgaggtt taaacgtcga cgcggccgct actagggaca ggattggtga
     2041 cagaaaagcc ccatccttag gcctcctcct tcctagtctc ctgatattgg gtctaacccc
     2101 cacctcctgt taggcagatt ccttatctgg tgacacaccc ccatttcctg gagccatctc
     2161 tctccttgcc agaacctcta aggtttgctt acgatggagc cagagaggat cctgggaggg
     2221 agagcttggc agggggtggg agggaagggg gggatgcgtg acctgcccgg ttctcagtgg
     2281 ccaccctgcg ctaccctctc ccagaacctg agctgctctg acgcggctgt ctggtgcgtt
     2341 tcactgatcc tggtgctgca gcttccttac acttcccaag aggagaagca gtttggaaaa
     2401 acaaaatcag aataagttgg tcctgagttc taactttggc tcttcacctt tctagtcccc
     2461 aatttatatt gttcctccgt gcgtcagttt tacctgtgag ataaggccag tagccagccc
     2521 cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt ggaaaactcc ctttgtgaga
     2581 atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct actccctttc tctttctcca
     2641 tccttctttc cttaaagagt ccccagtgct atctgggaca tattcctccg cccagagcag
     2701 ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt tgagtccttg gcaagcccag
     2761 gagaggcgct caggcttccc tgtccccctt cctcgtccac catctcatgc ccctggctct
     2821 cctgcccctt ccctacaggg gttcctggct ctgctctcca ccgcggtgga gctccagctt
     2881 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2941 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3001 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3061 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3121 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3181 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3241 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3301 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3361 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3421 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3481 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3541 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3601 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3661 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3721 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3781 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3841 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3901 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3961 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4021 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4081 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4141 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4201 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4261 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4321 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4381 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4441 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4501 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4561 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4621 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4681 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4741 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4801 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4861 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4921 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4981 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5041 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     5101 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     5161 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5221 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     5281 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     5341 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5401 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5461 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     5521 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     5581 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     5641 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg
     5701 taatacgact cactataggg cgaattgggt acctgctttc tctgaccagc attctctccc
     5761 ctgggcctgt gccgctttct gtctgcagct tgtggcctgg gtcacctcta cggctggccc
     5821 agatccttcc ctgccgcctc cttcaggttc cgtcttcctc cactccctct tccccttgct
     5881 ctctgctgtg ttgctgccca aggatgctct ttccggagca cttccttctc ggcgctgcac
     5941 cacgtgatgt cctctgagcg gatcctcccc gtgtctgggt cctctccggg catctctcct
     6001 ccctcaccca accccatgcc gtcttcactc gctgggttcc cttttccttc tccttctggg
     6061 gcctgtgcca tctctcgttt cttaggatgg ccttctccga cggatgtctc ccttgcgtcc
     6121 cgcctcccct tcttgtaggc ctgcatcatc accgtttttc tggacaaccc caaagtaccc
     6181 cgtctccctg gctttagcca cctctccatc ctcttgcttt ctttgcctgg acaccccgtt
     6241 ctcctgtgga ttcgggtcac ctctcactcc tttcatttgg gcagctcccc tacccccctt
     6301 acctctctag tctgtgctag ctcttccagc cccctgtcat ggcatcttcc aggggtccga
     6361 gagctcagct agtcttcttc ctccaacccg ggcccctatg tccacttcag gacagcatgt
     6421 ttgctgcctc cagggatcct gtgtccccga gctgggacca ccttatattc ccagggccgg
     6481 ttaatgtggc tctggttctg ggtactttta tctgtcccct ccaccccaca gtggggcaag
     6541 cttattaccc tgttatccct aaccggtgcg tttttatgct tgtagtattg tataatgttt
     6601 ttaagatcca agcttctgac ctcttctctt cctcccacag ggcctcgaga caattgatta
     6661 acatcgatac ggtaccgagt tggcgcgcct gggagctcac ggggacagcc cccccccaaa
     6721 gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg
     6781 ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc
     6841 gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag
     6901 cctgcagaca cctgggggga tacggggaaa aagctttagg ctgaaagaga gatttagaat
     6961 gacaggcgcg cctggccata catcgatacg gtaccgagtt ggcgcgcctg ggagctcacg
     7021 gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc
     7081 agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg
     7141 cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg
     7201 cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa agctttaggc
     7261 tgaaagagag atttagaatg acaggcgcgc ctggtggcga acgat
//
