LOCUS       40924_42139        7305 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT1_T1_100bp-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  JOURNAL   Unpublished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            "Unpublished"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..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    253..273
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             315..332
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             333..1028
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1071..1095)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1250..1305
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1666..1682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1690..1706
                     /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(1714..1744)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1759..1780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    1953..2789
                     /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(2819..2837)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2858..2874)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2882..2898)
                     /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(2906..2936)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2951..2972)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3260..3848)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4022..4879)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4880..4984)
                     /label=AmpR promoter
     rep_origin      complement(5010..5465)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5607..5623
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5633..5651
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5666..6469
                     /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    6548..6573
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    7238..7258
                     /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 tgttggttcg agtacgactc actataggat
      241 ccaattcgac cccaagtttg tacaaaaaag cagggatccc ggtaccctcg acacctgctg
      301 acggagccac catgcaccat caccatcacc atatggtgag cgagctgatt aaggagaaca
      361 tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag tgcacatccg
      421 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggcg gtcgagggcg
      481 gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc agcaaaacct
      541 tcatcaacca cacccagggc atccccgact tctttaagca gtccttcccc gagggcttca
      601 catgggagag agtcaccaca tacgaagatg ggggcgtgct gaccgctacc caggacacca
      661 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcccatcca
      721 acggccctgt gatgcagaag aaaacactcg gctgggaggc ctccaccgag acactgtacc
      781 ccgctgacgg cggcctggaa ggcagagccg acatggccct gaagctcgtg ggcgggggcc
      841 acctgatctg caaccttaag accacataca gatccaagaa acccgctaag aacctcaaga
      901 tgcccggcgt ctactatgtg gacaggagac tggaaagaat caaggaggcc gacaaagaga
      961 catacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg
     1021 ggcacaaact taattcctag taacgctcag tgcaggtgaa ttctaccacc acccagcttt
     1081 cttgtacaaa gtggtacgcg tgaattcact cctcaggtgc aggctgccta tcagaaggtg
     1141 gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc
     1201 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     1261 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     1321 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     1381 gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa
     1441 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     1501 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     1561 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     1621 ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc
     1681 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     1741 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     1801 cgctttccag tcgggaaacc tgtcgtgcca gcggatcgac agtactaagc ttggtgaatt
     1861 cccttatgtg agtgtaaaag gcaggcgagt ttgtccctta attaataggg ataacagggt
     1921 aatctcgagg tttaaacgtc gacgcggccg ctactaggga caggattggt gacagaaaag
     1981 ccccatcctt aggcctcctc cttcctagtc tcctgatatt gggtctaacc cccacctcct
     2041 gttaggcaga ttccttatct ggtgacacac ccccatttcc tggagccatc tctctccttg
     2101 ccagaacctc taaggtttgc ttacgatgga gccagagagg atcctgggag ggagagcttg
     2161 gcagggggtg ggagggaagg gggggatgcg tgacctgccc ggttctcagt ggccaccctg
     2221 cgctaccctc tcccagaacc tgagctgctc tgacgcggct gtctggtgcg tttcactgat
     2281 cctggtgctg cagcttcctt acacttccca agaggagaag cagtttggaa aaacaaaatc
     2341 agaataagtt ggtcctgagt tctaactttg gctcttcacc tttctagtcc ccaatttata
     2401 ttgttcctcc gtgcgtcagt tttacctgtg agataaggcc agtagccagc cccgtcctgg
     2461 cagggctgtg gtgaggaggg gggtgtccgt gtggaaaact ccctttgtga gaatggtgcg
     2521 tcctaggtgt tcaccaggtc gtggccgcct ctactccctt tctctttctc catccttctt
     2581 tccttaaaga gtccccagtg ctatctggga catattcctc cgcccagagc agggtcccgc
     2641 ttccctaagg ccctgctctg ggcttctggg tttgagtcct tggcaagccc aggagaggcg
     2701 ctcaggcttc cctgtccccc ttcctcgtcc accatctcat gcccctggct ctcctgcccc
     2761 ttccctacag gggttcctgg ctctgctctc caccgcggtg gagctccagc ttttgttccc
     2821 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2881 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2941 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3001 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3061 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3121 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3181 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3241 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3301 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3361 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3421 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3481 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3541 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3601 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3661 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3721 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3781 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3841 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3901 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3961 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4021 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4081 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4141 gtgctgcaat gataccgcga gagccacgct caccggctcc agatttatca gcaataaacc
     4201 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4261 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4321 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4381 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4441 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4501 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4561 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4621 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4681 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4741 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4801 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4861 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4921 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4981 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     5041 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     5101 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     5161 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     5221 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     5281 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     5341 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     5401 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     5461 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     5521 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     5581 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga
     5641 ctcactatag ggcgaattgg gtacctgctt tctctgacca gcattctctc ccctgggcct
     5701 gtgccgcttt ctgtctgcag cttgtggcct gggtcacctc tacggctggc ccagatcctt
     5761 ccctgccgcc tccttcaggt tccgtcttcc tccactccct cttccccttg ctctctgctg
     5821 tgttgctgcc caaggatgct ctttccggag cacttccttc tcggcgctgc accacgtgat
     5881 gtcctctgag cggatcctcc ccgtgtctgg gtcctctccg ggcatctctc ctccctcacc
     5941 caaccccatg ccgtcttcac tcgctgggtt cccttttcct tctccttctg gggcctgtgc
     6001 catctctcgt ttcttaggat ggccttctcc gacggatgtc tcccttgcgt cccgcctccc
     6061 cttcttgtag gcctgcatca tcaccgtttt tctggacaac cccaaagtac cccgtctccc
     6121 tggctttagc cacctctcca tcctcttgct ttctttgcct ggacaccccg ttctcctgtg
     6181 gattcgggtc acctctcact cctttcattt gggcagctcc cctacccccc ttacctctct
     6241 agtctgtgct agctcttcca gccccctgtc atggcatctt ccaggggtcc gagagctcag
     6301 ctagtcttct tcctccaacc cgggccccta tgtccacttc aggacagcat gtttgctgcc
     6361 tccagggatc ctgtgtcccc gagctgggac caccttatat tcccagggcc ggttaatgtg
     6421 gctctggttc tgggtacttt tatctgtccc ctccacccca cagtggggca agcttattac
     6481 cctgttatcc ctaaccggtc taatacccaa tctctcgtct tatccagatg ttttatacgc
     6541 caagcttctg acctcttctc ttcctcccac agggcctcga gacaattgat taacatcgat
     6601 acggtaccga gttggcgcgc ctgggagctc acggggacag ccccccccca aagcccccag
     6661 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     6721 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     6781 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     6841 cacctggggg gatacgggga aaaagcttta ggctgaaaga gagatttaga atgacaggcg
     6901 cgcctggcca tacatcgata cggtaccgag ttggcgcgcc tgggagctca cggggacagc
     6961 ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga
     7021 gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc
     7081 ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc
     7141 tgctctttga gcctgcagac acctgggggg atacggggaa aaagctttag gctgaaagag
     7201 agatttagaa tgacaggcgc gcctggtggc gaacgatcaa ctttgtatag aaaagttggc
     7261 tccgaatttc tcgacagatc tcatgtgatt acgccaagct acggg
//
