LOCUS       40924_42399        7333 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT9_T9x3_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7333)
  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  JOURNAL   Unpublished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 7333)
  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 7333)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7333)
  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            "Unpublished"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..7333
                     /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..197
                     /label=TALER9 bs binding site
                     /bound_moiety="TALER9 bs"
     misc_feature    204..263
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    270..290
                     /label=TALER9 bs binding site
                     /bound_moiety="TALER9 bs"
     protein_bind    291..311
                     /label=TALER9 bs binding site
                     /bound_moiety="TALER9 bs"
     protein_bind    312..332
                     /label=TALER9 bs binding site
                     /bound_moiety="TALER9 bs"
     protein_bind    349..369
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             411..428
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             429..1124
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1167..1191)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1346..1401
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1762..1778)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1786..1802
                     /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(1810..1840)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1855..1876
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2049..2885
                     /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(2915..2933)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2954..2970)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2978..2994)
                     /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(3002..3032)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3047..3068)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3356..3944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4118..4975)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4976..5080)
                     /label=AmpR promoter
     rep_origin      complement(5106..5561)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5703..5719
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5729..5747
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5762..6565
                     /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    6644..6669
                     /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 tctagatata
      181 gaaccgatcc tcccattgaa ttctaggcgt gtacggtggg aggcctatat aagcagagct
      241 cgtttagtga accgtcagat cgcctcgagt atagaaccga tcctcccatt tatagaaccg
      301 atcctcccat ttatagaacc gatcctccca ttggatccaa ttcgacccca agtttgtaca
      361 aaaaagcagg gatcccggta ccctcgacac ctgctgacgg agccaccatg caccatcacc
      421 atcaccatat ggtgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg
      481 gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg
      541 gcacccagac catgagaatc aaggcggtcg agggcggccc tctccccttc gccttcgaca
      601 tcctggctac cagcttcatg tacggcagca aaaccttcat caaccacacc cagggcatcc
      661 ccgacttctt taagcagtcc ttccccgagg gcttcacatg ggagagagtc accacatacg
      721 aagatggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct
      781 acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg cagaagaaaa
      841 cactcggctg ggaggcctcc accgagacac tgtaccccgc tgacggcggc ctggaaggca
      901 gagccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac cttaagacca
      961 catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac tatgtggaca
     1021 ggagactgga aagaatcaag gaggccgaca aagagacata cgtcgagcag cacgaggtgg
     1081 ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat tcctagtaac
     1141 gctcagtgca ggtgaattct accaccaccc agctttcttg tacaaagtgg tacgcgtgaa
     1201 ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     1261 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     1321 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     1381 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     1441 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     1501 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     1561 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     1621 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     1681 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     1741 cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     1801 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     1861 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1921 gtgccagcgg atcgacagta ctaagcttgg tctaataccc aatctctcgt cttatccaga
     1981 tgttttatac gccttaatta atagggataa cagggtaatc tcgaggttta aacgtcgacg
     2041 cggccgctac tagggacagg attggtgaca gaaaagcccc atccttaggc ctcctccttc
     2101 ctagtctcct gatattgggt ctaaccccca cctcctgtta ggcagattcc ttatctggtg
     2161 acacaccccc atttcctgga gccatctctc tccttgccag aacctctaag gtttgcttac
     2221 gatggagcca gagaggatcc tgggagggag agcttggcag ggggtgggag ggaagggggg
     2281 gatgcgtgac ctgcccggtt ctcagtggcc accctgcgct accctctccc agaacctgag
     2341 ctgctctgac gcggctgtct ggtgcgtttc actgatcctg gtgctgcagc ttccttacac
     2401 ttcccaagag gagaagcagt ttggaaaaac aaaatcagaa taagttggtc ctgagttcta
     2461 actttggctc ttcacctttc tagtccccaa tttatattgt tcctccgtgc gtcagtttta
     2521 cctgtgagat aaggccagta gccagccccg tcctggcagg gctgtggtga ggaggggggt
     2581 gtccgtgtgg aaaactccct ttgtgagaat ggtgcgtcct aggtgttcac caggtcgtgg
     2641 ccgcctctac tccctttctc tttctccatc cttctttcct taaagagtcc ccagtgctat
     2701 ctgggacata ttcctccgcc cagagcaggg tcccgcttcc ctaaggccct gctctgggct
     2761 tctgggtttg agtccttggc aagcccagga gaggcgctca ggcttccctg tcccccttcc
     2821 tcgtccacca tctcatgccc ctggctctcc tgccccttcc ctacaggggt tcctggctct
     2881 gctctccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     2941 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3001 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3061 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3121 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3181 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3241 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3301 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3361 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3421 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3481 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3541 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3601 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3661 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3721 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3781 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3841 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3901 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3961 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4021 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4081 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4141 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4201 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4261 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4321 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4381 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4441 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4501 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4561 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4621 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4681 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4741 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4801 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4861 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4921 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4981 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5041 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5101 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5161 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5221 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5281 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5341 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5401 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5461 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     5521 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     5581 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     5641 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     5701 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
     5761 ctgctttctc tgaccagcat tctctcccct gggcctgtgc cgctttctgt ctgcagcttg
     5821 tggcctgggt cacctctacg gctggcccag atccttccct gccgcctcct tcaggttccg
     5881 tcttcctcca ctccctcttc cccttgctct ctgctgtgtt gctgcccaag gatgctcttt
     5941 ccggagcact tccttctcgg cgctgcacca cgtgatgtcc tctgagcgga tcctccccgt
     6001 gtctgggtcc tctccgggca tctctcctcc ctcacccaac cccatgccgt cttcactcgc
     6061 tgggttccct tttccttctc cttctggggc ctgtgccatc tctcgtttct taggatggcc
     6121 ttctccgacg gatgtctccc ttgcgtcccg cctccccttc ttgtaggcct gcatcatcac
     6181 cgtttttctg gacaacccca aagtaccccg tctccctggc tttagccacc tctccatcct
     6241 cttgctttct ttgcctggac accccgttct cctgtggatt cgggtcacct ctcactcctt
     6301 tcatttgggc agctccccta ccccccttac ctctctagtc tgtgctagct cttccagccc
     6361 cctgtcatgg catcttccag gggtccgaga gctcagctag tcttcttcct ccaacccggg
     6421 cccctatgtc cacttcagga cagcatgttt gctgcctcca gggatcctgt gtccccgagc
     6481 tgggaccacc ttatattccc agggccggtt aatgtggctc tggttctggg tacttttatc
     6541 tgtcccctcc accccacagt ggggcaagct tattaccctg ttatccctaa ccggtgcgtt
     6601 tttatgcttg tagtattgta taatgttttt aagatccaag cttctgacct cttctcttcc
     6661 tcccacaggg cctcgagaca attgattaac atcgatacgg taccgagttg gcgcgcctgg
     6721 gagctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg
     6781 ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc
     6841 ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc
     6901 ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tggggggata cggggaaaaa
     6961 gctttaggct gaaagagaga tttagaatga caggcgcgcc tggccataca tcgatacggt
     7021 accgagttgg cgcgcctggg agctcacggg gacagccccc ccccaaagcc cccagggatg
     7081 taattacgtc cctcccccgc tagggggcag cagcgagccg cccggggctc cgctccggtc
     7141 cggcgctccc cccgcatccc cgagccggca gcgtgcgggg acagcccggg cacggggaag
     7201 gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct
     7261 ggggggatac ggggaaaaag ctttaggctg aaagagagat ttagaatgac aggcgcgcct
     7321 ggtggcgaac gat
//
