LOCUS       40924_42079        7317 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT14_T14x3_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7317)
  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  JOURNAL   Unpublished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 7317)
  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 7317)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7317)
  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            "Unpublished"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..7317
                     /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..193
                     /label=TALER14 bs binding site
                     /bound_moiety="TALER14 bs"
     misc_feature    200..259
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    266..282
                     /label=TALER14 bs binding site
                     /bound_moiety="TALER14 bs"
     protein_bind    283..299
                     /label=TALER14 bs binding site
                     /bound_moiety="TALER14 bs"
     protein_bind    300..316
                     /label=TALER14 bs binding site
                     /bound_moiety="TALER14 bs"
     protein_bind    333..353
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             395..412
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             413..1108
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1151..1175)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1330..1385
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1746..1762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1770..1786
                     /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(1794..1824)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1839..1860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2033..2869
                     /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(2899..2917)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2938..2954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2962..2978)
                     /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(2986..3016)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3031..3052)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3340..3928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4102..4959)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4960..5064)
                     /label=AmpR promoter
     rep_origin      complement(5090..5545)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5687..5703
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5713..5731
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5746..6549
                     /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    6628..6653
                     /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 tctagatacc
      181 cctctccgct tctgaattct aggcgtgtac ggtgggaggc ctatataagc agagctcgtt
      241 tagtgaaccg tcagatcgcc tcgagtaccc ctctccgctt cttacccctc tccgcttctt
      301 acccctctcc gcttctggat ccaattcgac cccaagtttg tacaaaaaag cagggatccc
      361 ggtaccctcg acacctgctg acggagccac catgcaccat caccatcacc atatggtgag
      421 cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tgaacaacca
      481 ccacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag
      541 aatcaaggcg gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt
      601 catgtacggc agcaaaacct tcatcaacca cacccagggc atccccgact tctttaagca
      661 gtccttcccc gagggcttca catgggagag agtcaccaca tacgaagatg ggggcgtgct
      721 gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag
      781 aggggtgaac ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc
      841 ctccaccgag acactgtacc ccgctgacgg cggcctggaa ggcagagccg acatggccct
      901 gaagctcgtg ggcgggggcc acctgatctg caaccttaag accacataca gatccaagaa
      961 acccgctaag aacctcaaga tgcccggcgt ctactatgtg gacaggagac tggaaagaat
     1021 caaggaggcc gacaaagaga catacgtcga gcagcacgag gtggctgtgg ccagatactg
     1081 cgacctccct agcaaactgg ggcacaaact taattcctag taacgctcag tgcaggtgaa
     1141 ttctaccacc acccagcttt cttgtacaaa gtggtacgcg tgaattcact cctcaggtgc
     1201 aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact
     1261 gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga
     1321 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
     1381 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg
     1441 tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa
     1501 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
     1561 tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc
     1621 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
     1681 tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa
     1741 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     1801 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     1861 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatcgac
     1921 agtactaagc ttggtctaat acccaatctc tcgtcttatc cagatgtttt atacgcctta
     1981 attaataggg ataacagggt aatctcgagg tttaaacgtc gacgcggccg ctactaggga
     2041 caggattggt gacagaaaag ccccatcctt aggcctcctc cttcctagtc tcctgatatt
     2101 gggtctaacc cccacctcct gttaggcaga ttccttatct ggtgacacac ccccatttcc
     2161 tggagccatc tctctccttg ccagaacctc taaggtttgc ttacgatgga gccagagagg
     2221 atcctgggag ggagagcttg gcagggggtg ggagggaagg gggggatgcg tgacctgccc
     2281 ggttctcagt ggccaccctg cgctaccctc tcccagaacc tgagctgctc tgacgcggct
     2341 gtctggtgcg tttcactgat cctggtgctg cagcttcctt acacttccca agaggagaag
     2401 cagtttggaa aaacaaaatc agaataagtt ggtcctgagt tctaactttg gctcttcacc
     2461 tttctagtcc ccaatttata ttgttcctcc gtgcgtcagt tttacctgtg agataaggcc
     2521 agtagccagc cccgtcctgg cagggctgtg gtgaggaggg gggtgtccgt gtggaaaact
     2581 ccctttgtga gaatggtgcg tcctaggtgt tcaccaggtc gtggccgcct ctactccctt
     2641 tctctttctc catccttctt tccttaaaga gtccccagtg ctatctggga catattcctc
     2701 cgcccagagc agggtcccgc ttccctaagg ccctgctctg ggcttctggg tttgagtcct
     2761 tggcaagccc aggagaggcg ctcaggcttc cctgtccccc ttcctcgtcc accatctcat
     2821 gcccctggct ctcctgcccc ttccctacag gggttcctgg ctctgctctc caccgcggtg
     2881 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     2941 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3001 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3061 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3121 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3181 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3241 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3301 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3361 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3421 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3481 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3541 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3601 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3661 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3721 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3781 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3841 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3901 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3961 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4021 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4081 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4141 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4201 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4261 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4321 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4381 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4441 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4501 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4561 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4621 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4681 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4741 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4801 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4861 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4921 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4981 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5041 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     5101 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5161 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5221 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     5281 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5341 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5401 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     5461 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     5521 cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa
     5581 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     5641 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     5701 agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtacctgctt tctctgacca
     5761 gcattctctc ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct gggtcacctc
     5821 tacggctggc ccagatcctt ccctgccgcc tccttcaggt tccgtcttcc tccactccct
     5881 cttccccttg ctctctgctg tgttgctgcc caaggatgct ctttccggag cacttccttc
     5941 tcggcgctgc accacgtgat gtcctctgag cggatcctcc ccgtgtctgg gtcctctccg
     6001 ggcatctctc ctccctcacc caaccccatg ccgtcttcac tcgctgggtt cccttttcct
     6061 tctccttctg gggcctgtgc catctctcgt ttcttaggat ggccttctcc gacggatgtc
     6121 tcccttgcgt cccgcctccc cttcttgtag gcctgcatca tcaccgtttt tctggacaac
     6181 cccaaagtac cccgtctccc tggctttagc cacctctcca tcctcttgct ttctttgcct
     6241 ggacaccccg ttctcctgtg gattcgggtc acctctcact cctttcattt gggcagctcc
     6301 cctacccccc ttacctctct agtctgtgct agctcttcca gccccctgtc atggcatctt
     6361 ccaggggtcc gagagctcag ctagtcttct tcctccaacc cgggccccta tgtccacttc
     6421 aggacagcat gtttgctgcc tccagggatc ctgtgtcccc gagctgggac caccttatat
     6481 tcccagggcc ggttaatgtg gctctggttc tgggtacttt tatctgtccc ctccacccca
     6541 cagtggggca agcttattac cctgttatcc ctaaccggtg cgtttttatg cttgtagtat
     6601 tgtataatgt ttttaagatc caagcttctg acctcttctc ttcctcccac agggcctcga
     6661 gacaattgat taacatcgat acggtaccga gttggcgcgc ctgggagctc acggggacag
     6721 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     6781 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     6841 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     6901 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgaaaga
     6961 gagatttaga atgacaggcg cgcctggcca tacatcgata cggtaccgag ttggcgcgcc
     7021 tgggagctca cggggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc
     7081 ccgctagggg gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca
     7141 tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct
     7201 ttcctctgaa cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa
     7261 aaagctttag gctgaaagag agatttagaa tgacaggcgc gcctggtggc gaacgat
//
