LOCUS       40924_42049        7329 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT10_T10x3_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7329)
  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  JOURNAL   Unpublished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 7329)
  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 7329)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7329)
  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            "Unpublished"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..7329
                     /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..196
                     /label=TALER10 bs binding site
                     /bound_moiety="TALER10 bs"
     misc_feature    203..262
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    269..288
                     /label=TALER10 bs binding site
                     /bound_moiety="TALER10 bs"
     protein_bind    289..308
                     /label=TALER10 bs binding site
                     /bound_moiety="TALER10 bs"
     protein_bind    309..328
                     /label=TALER10 bs binding site
                     /bound_moiety="TALER10 bs"
     protein_bind    345..365
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             407..424
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             425..1120
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1163..1187)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1342..1397
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1758..1774)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1782..1798
                     /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(1806..1836)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1851..1872
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2045..2881
                     /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(2911..2929)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2950..2966)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2974..2990)
                     /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(2998..3028)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3043..3064)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3352..3940)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4114..4971)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4972..5076)
                     /label=AmpR promoter
     rep_origin      complement(5102..5557)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5699..5715
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5725..5743
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5758..6561
                     /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    6640..6665
                     /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 tcatcaggaa catgttgaat tctaggcgtg tacggtggga ggcctatata agcagagctc
      241 gtttagtgaa ccgtcagatc gcctcgagta cctcatcagg aacatgttta cctcatcagg
      301 aacatgttta cctcatcagg aacatgttgg atccaattcg accccaagtt tgtacaaaaa
      361 agcagggatc ccggtaccct cgacacctgc tgacggagcc accatgcacc atcaccatca
      421 ccatatggtg agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
      481 cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
      541 ccagaccatg agaatcaagg cggtcgaggg cggccctctc cccttcgcct tcgacatcct
      601 ggctaccagc ttcatgtacg gcagcaaaac cttcatcaac cacacccagg gcatccccga
      661 cttctttaag cagtccttcc ccgagggctt cacatgggag agagtcacca catacgaaga
      721 tgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
      781 cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact
      841 cggctgggag gcctccaccg agacactgta ccccgctgac ggcggcctgg aaggcagagc
      901 cgacatggcc ctgaagctcg tgggcggggg ccacctgatc tgcaacctta agaccacata
      961 cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg tggacaggag
     1021 actggaaaga atcaaggagg ccgacaaaga gacatacgtc gagcagcacg aggtggctgt
     1081 ggccagatac tgcgacctcc ctagcaaact ggggcacaaa cttaattcct agtaacgctc
     1141 agtgcaggtg aattctacca ccacccagct ttcttgtaca aagtggtacg cgtgaattca
     1201 ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc
     1261 acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc
     1321 ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga
     1381 attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga
     1441 atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag
     1501 gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc
     1561 atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt
     1621 ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct
     1681 gactactccc agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca
     1741 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1801 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1861 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1921 cagcggatcg acagtactaa gcttggtcta atacccaatc tctcgtctta tccagatgtt
     1981 ttatacgcct taattaatag ggataacagg gtaatctcga ggtttaaacg tcgacgcggc
     2041 cgctactagg gacaggattg gtgacagaaa agccccatcc ttaggcctcc tccttcctag
     2101 tctcctgata ttgggtctaa cccccacctc ctgttaggca gattccttat ctggtgacac
     2161 acccccattt cctggagcca tctctctcct tgccagaacc tctaaggttt gcttacgatg
     2221 gagccagaga ggatcctggg agggagagct tggcaggggg tgggagggaa gggggggatg
     2281 cgtgacctgc ccggttctca gtggccaccc tgcgctaccc tctcccagaa cctgagctgc
     2341 tctgacgcgg ctgtctggtg cgtttcactg atcctggtgc tgcagcttcc ttacacttcc
     2401 caagaggaga agcagtttgg aaaaacaaaa tcagaataag ttggtcctga gttctaactt
     2461 tggctcttca cctttctagt ccccaattta tattgttcct ccgtgcgtca gttttacctg
     2521 tgagataagg ccagtagcca gccccgtcct ggcagggctg tggtgaggag gggggtgtcc
     2581 gtgtggaaaa ctccctttgt gagaatggtg cgtcctaggt gttcaccagg tcgtggccgc
     2641 ctctactccc tttctctttc tccatccttc tttccttaaa gagtccccag tgctatctgg
     2701 gacatattcc tccgcccaga gcagggtccc gcttccctaa ggccctgctc tgggcttctg
     2761 ggtttgagtc cttggcaagc ccaggagagg cgctcaggct tccctgtccc ccttcctcgt
     2821 ccaccatctc atgcccctgg ctctcctgcc ccttccctac aggggttcct ggctctgctc
     2881 tccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     2941 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3001 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3061 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3121 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3181 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3241 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3301 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3361 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3421 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3481 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3541 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3601 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3661 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3721 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3781 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3841 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3901 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3961 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4021 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4081 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4141 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4201 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4261 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4321 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4381 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4441 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4501 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4561 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4621 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4681 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4741 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4801 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4861 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4921 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4981 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5041 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5101 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5161 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5221 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     5281 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     5341 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5401 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     5461 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     5521 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     5581 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     5641 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     5701 aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtacctgc
     5761 tttctctgac cagcattctc tcccctgggc ctgtgccgct ttctgtctgc agcttgtggc
     5821 ctgggtcacc tctacggctg gcccagatcc ttccctgccg cctccttcag gttccgtctt
     5881 cctccactcc ctcttcccct tgctctctgc tgtgttgctg cccaaggatg ctctttccgg
     5941 agcacttcct tctcggcgct gcaccacgtg atgtcctctg agcggatcct ccccgtgtct
     6001 gggtcctctc cgggcatctc tcctccctca cccaacccca tgccgtcttc actcgctggg
     6061 ttcccttttc cttctccttc tggggcctgt gccatctctc gtttcttagg atggccttct
     6121 ccgacggatg tctcccttgc gtcccgcctc cccttcttgt aggcctgcat catcaccgtt
     6181 tttctggaca accccaaagt accccgtctc cctggcttta gccacctctc catcctcttg
     6241 ctttctttgc ctggacaccc cgttctcctg tggattcggg tcacctctca ctcctttcat
     6301 ttgggcagct cccctacccc ccttacctct ctagtctgtg ctagctcttc cagccccctg
     6361 tcatggcatc ttccaggggt ccgagagctc agctagtctt cttcctccaa cccgggcccc
     6421 tatgtccact tcaggacagc atgtttgctg cctccaggga tcctgtgtcc ccgagctggg
     6481 accaccttat attcccaggg ccggttaatg tggctctggt tctgggtact tttatctgtc
     6541 ccctccaccc cacagtgggg caagcttatt accctgttat ccctaaccgg tgcgttttta
     6601 tgcttgtagt attgtataat gtttttaaga tccaagcttc tgacctcttc tcttcctccc
     6661 acagggcctc gagacaattg attaacatcg atacggtacc gagttggcgc gcctgggagc
     6721 tcacggggac agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag
     6781 ggggcagcag cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga
     6841 gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct
     6901 gaacgcttct cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaagctt
     6961 taggctgaaa gagagattta gaatgacagg cgcgcctggc catacatcga tacggtaccg
     7021 agttggcgcg cctgggagct cacggggaca gccccccccc aaagccccca gggatgtaat
     7081 tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc
     7141 gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg
     7201 cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg
     7261 ggatacgggg aaaaagcttt aggctgaaag agagatttag aatgacaggc gcgcctggtg
     7321 gcgaacgat
//
