LOCUS       40924_42289        7277 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT32_T32_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7277)
  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  JOURNAL   Unpublished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 7277)
  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 7277)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7277)
  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            "Unpublished"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..7277
                     /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    293..313
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             355..372
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             373..1068
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1111..1135)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1290..1345
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1706..1722)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1730..1746
                     /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(1754..1784)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1799..1820
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    1993..2829
                     /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(2859..2877)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2898..2914)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2922..2938)
                     /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(2946..2976)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2991..3012)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3300..3888)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4062..4919)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4920..5024)
                     /label=AmpR promoter
     rep_origin      complement(5050..5505)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5647..5663
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5673..5691
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5706..6509
                     /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    6588..6613
                     /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 actataggat ccaattcgac cccaagtttg
      301 tacaaaaaag cagggatccc ggtaccctcg acacctgctg acggagccac catgcaccat
      361 caccatcacc atatggtgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg
      421 gagggcaccg tgaacaacca ccacttcaag tgcacatccg agggcgaagg caagccctac
      481 gagggcaccc agaccatgag aatcaaggcg gtcgagggcg gccctctccc cttcgccttc
      541 gacatcctgg ctaccagctt catgtacggc agcaaaacct tcatcaacca cacccagggc
      601 atccccgact tctttaagca gtccttcccc gagggcttca catgggagag agtcaccaca
      661 tacgaagatg ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc
      721 atctacaacg tcaagatcag aggggtgaac ttcccatcca acggccctgt gatgcagaag
      781 aaaacactcg gctgggaggc ctccaccgag acactgtacc ccgctgacgg cggcctggaa
      841 ggcagagccg acatggccct gaagctcgtg ggcgggggcc acctgatctg caaccttaag
      901 accacataca gatccaagaa acccgctaag aacctcaaga tgcccggcgt ctactatgtg
      961 gacaggagac tggaaagaat caaggaggcc gacaaagaga catacgtcga gcagcacgag
     1021 gtggctgtgg ccagatactg cgacctccct agcaaactgg ggcacaaact taattcctag
     1081 taacgctcag tgcaggtgaa ttctaccacc acccagcttt cttgtacaaa gtggtacgcg
     1141 tgaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc
     1201 cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat
     1261 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     1321 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     1381 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat
     1441 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     1501 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     1561 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     1621 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct
     1681 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1741 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1801 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1861 tgtcgtgcca gcggatcgac agtactaagc ttggtctaat acccaatctc tcgtcttatc
     1921 cagatgtttt atacgcctta attaataggg ataacagggt aatctcgagg tttaaacgtc
     1981 gacgcggccg ctactaggga caggattggt gacagaaaag ccccatcctt aggcctcctc
     2041 cttcctagtc tcctgatatt gggtctaacc cccacctcct gttaggcaga ttccttatct
     2101 ggtgacacac ccccatttcc tggagccatc tctctccttg ccagaacctc taaggtttgc
     2161 ttacgatgga gccagagagg atcctgggag ggagagcttg gcagggggtg ggagggaagg
     2221 gggggatgcg tgacctgccc ggttctcagt ggccaccctg cgctaccctc tcccagaacc
     2281 tgagctgctc tgacgcggct gtctggtgcg tttcactgat cctggtgctg cagcttcctt
     2341 acacttccca agaggagaag cagtttggaa aaacaaaatc agaataagtt ggtcctgagt
     2401 tctaactttg gctcttcacc tttctagtcc ccaatttata ttgttcctcc gtgcgtcagt
     2461 tttacctgtg agataaggcc agtagccagc cccgtcctgg cagggctgtg gtgaggaggg
     2521 gggtgtccgt gtggaaaact ccctttgtga gaatggtgcg tcctaggtgt tcaccaggtc
     2581 gtggccgcct ctactccctt tctctttctc catccttctt tccttaaaga gtccccagtg
     2641 ctatctggga catattcctc cgcccagagc agggtcccgc ttccctaagg ccctgctctg
     2701 ggcttctggg tttgagtcct tggcaagccc aggagaggcg ctcaggcttc cctgtccccc
     2761 ttcctcgtcc accatctcat gcccctggct ctcctgcccc ttccctacag gggttcctgg
     2821 ctctgctctc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg
     2881 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2941 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3001 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3061 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3121 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3181 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3241 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3301 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3361 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3421 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3481 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3541 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3601 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3661 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3721 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3781 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3841 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3901 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3961 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4021 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4081 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4141 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4201 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4261 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4321 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4381 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4441 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4501 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4561 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4621 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4681 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4741 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4801 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4861 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4921 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4981 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5041 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     5101 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     5161 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     5221 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     5281 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     5341 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     5401 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     5461 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca
     5521 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     5581 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     5641 gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg
     5701 gtacctgctt tctctgacca gcattctctc ccctgggcct gtgccgcttt ctgtctgcag
     5761 cttgtggcct gggtcacctc tacggctggc ccagatcctt ccctgccgcc tccttcaggt
     5821 tccgtcttcc tccactccct cttccccttg ctctctgctg tgttgctgcc caaggatgct
     5881 ctttccggag cacttccttc tcggcgctgc accacgtgat gtcctctgag cggatcctcc
     5941 ccgtgtctgg gtcctctccg ggcatctctc ctccctcacc caaccccatg ccgtcttcac
     6001 tcgctgggtt cccttttcct tctccttctg gggcctgtgc catctctcgt ttcttaggat
     6061 ggccttctcc gacggatgtc tcccttgcgt cccgcctccc cttcttgtag gcctgcatca
     6121 tcaccgtttt tctggacaac cccaaagtac cccgtctccc tggctttagc cacctctcca
     6181 tcctcttgct ttctttgcct ggacaccccg ttctcctgtg gattcgggtc acctctcact
     6241 cctttcattt gggcagctcc cctacccccc ttacctctct agtctgtgct agctcttcca
     6301 gccccctgtc atggcatctt ccaggggtcc gagagctcag ctagtcttct tcctccaacc
     6361 cgggccccta tgtccacttc aggacagcat gtttgctgcc tccagggatc ctgtgtcccc
     6421 gagctgggac caccttatat tcccagggcc ggttaatgtg gctctggttc tgggtacttt
     6481 tatctgtccc ctccacccca cagtggggca agcttattac cctgttatcc ctaaccggtg
     6541 cgtttttatg cttgtagtat tgtataatgt ttttaagatc caagcttctg acctcttctc
     6601 ttcctcccac agggcctcga gacaattgat taacatcgat acggtaccga gttggcgcgc
     6661 ctgggagctc acggggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc
     6721 cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc
     6781 atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc
     6841 tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga
     6901 aaaagcttta ggctgaaaga gagatttaga atgacaggcg cgcctggcca tacatcgata
     6961 cggtaccgag ttggcgcgcc tgggagctca cggggacagc ccccccccaa agcccccagg
     7021 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     7081 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     7141 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     7201 acctgggggg atacggggaa aaagctttag gctgaaagag agatttagaa tgacaggcgc
     7261 gcctggtggc gaacgat
//
