LOCUS       40924_42209        7287 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT22_T22_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7287)
  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  JOURNAL   Unpublished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 7287)
  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 7287)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7287)
  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            "Unpublished"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..7287
                     /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..195
                     /label=TALER22 bs binding site
                     /bound_moiety="TALER22 bs"
     misc_feature    202..261
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    268..286
                     /label=TALER22 bs binding site
                     /bound_moiety="TALER22 bs"
     protein_bind    303..323
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             365..382
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             383..1078
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1121..1145)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1300..1355
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1716..1732)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1740..1756
                     /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(1764..1794)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1809..1830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2003..2839
                     /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(2869..2887)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2908..2924)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2932..2948)
                     /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(2956..2986)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3001..3022)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3310..3898)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4072..4929)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4930..5034)
                     /label=AmpR promoter
     rep_origin      complement(5060..5515)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5657..5673
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5683..5701
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5716..6519
                     /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    6598..6623
                     /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 tctagatagt
      181 cagcaccagg catttgaatt ctaggcgtgt acggtgggag gcctatataa gcagagctcg
      241 tttagtgaac cgtcagatcg cctcgagtag tcagcaccag gcatttggat ccaattcgac
      301 cccaagtttg tacaaaaaag cagggatccc ggtaccctcg acacctgctg acggagccac
      361 catgcaccat caccatcacc atatggtgag cgagctgatt aaggagaaca tgcacatgaa
      421 gctgtacatg gagggcaccg tgaacaacca ccacttcaag tgcacatccg agggcgaagg
      481 caagccctac gagggcaccc agaccatgag aatcaaggcg gtcgagggcg gccctctccc
      541 cttcgccttc gacatcctgg ctaccagctt catgtacggc agcaaaacct tcatcaacca
      601 cacccagggc atccccgact tctttaagca gtccttcccc gagggcttca catgggagag
      661 agtcaccaca tacgaagatg ggggcgtgct gaccgctacc caggacacca gcctccagga
      721 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcccatcca acggccctgt
      781 gatgcagaag aaaacactcg gctgggaggc ctccaccgag acactgtacc ccgctgacgg
      841 cggcctggaa ggcagagccg acatggccct gaagctcgtg ggcgggggcc acctgatctg
      901 caaccttaag accacataca gatccaagaa acccgctaag aacctcaaga tgcccggcgt
      961 ctactatgtg gacaggagac tggaaagaat caaggaggcc gacaaagaga catacgtcga
     1021 gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg ggcacaaact
     1081 taattcctag taacgctcag tgcaggtgaa ttctaccacc acccagcttt cttgtacaaa
     1141 gtggtacgcg tgaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     1201 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg
     1261 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     1321 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     1381 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc
     1441 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg
     1501 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt
     1561 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca
     1621 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat
     1681 ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     1741 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     1801 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1861 tcgggaaacc tgtcgtgcca gcggatcgac agtactaagc ttggtctaat acccaatctc
     1921 tcgtcttatc cagatgtttt atacgcctta attaataggg ataacagggt aatctcgagg
     1981 tttaaacgtc gacgcggccg ctactaggga caggattggt gacagaaaag ccccatcctt
     2041 aggcctcctc cttcctagtc tcctgatatt gggtctaacc cccacctcct gttaggcaga
     2101 ttccttatct ggtgacacac ccccatttcc tggagccatc tctctccttg ccagaacctc
     2161 taaggtttgc ttacgatgga gccagagagg atcctgggag ggagagcttg gcagggggtg
     2221 ggagggaagg gggggatgcg tgacctgccc ggttctcagt ggccaccctg cgctaccctc
     2281 tcccagaacc tgagctgctc tgacgcggct gtctggtgcg tttcactgat cctggtgctg
     2341 cagcttcctt acacttccca agaggagaag cagtttggaa aaacaaaatc agaataagtt
     2401 ggtcctgagt tctaactttg gctcttcacc tttctagtcc ccaatttata ttgttcctcc
     2461 gtgcgtcagt tttacctgtg agataaggcc agtagccagc cccgtcctgg cagggctgtg
     2521 gtgaggaggg gggtgtccgt gtggaaaact ccctttgtga gaatggtgcg tcctaggtgt
     2581 tcaccaggtc gtggccgcct ctactccctt tctctttctc catccttctt tccttaaaga
     2641 gtccccagtg ctatctggga catattcctc cgcccagagc agggtcccgc ttccctaagg
     2701 ccctgctctg ggcttctggg tttgagtcct tggcaagccc aggagaggcg ctcaggcttc
     2761 cctgtccccc ttcctcgtcc accatctcat gcccctggct ctcctgcccc ttccctacag
     2821 gggttcctgg ctctgctctc caccgcggtg gagctccagc ttttgttccc tttagtgagg
     2881 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2941 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3001 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3061 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3121 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3181 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3241 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3301 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3361 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3421 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3481 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3541 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3601 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3661 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3721 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3781 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3841 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3901 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3961 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4021 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4081 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4141 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4201 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4261 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4321 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4381 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4441 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4501 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4561 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4621 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4681 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4741 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4801 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4861 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4921 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4981 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5041 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5101 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5161 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5221 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5281 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5341 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5401 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5461 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     5521 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     5581 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     5641 tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag
     5701 ggcgaattgg gtacctgctt tctctgacca gcattctctc ccctgggcct gtgccgcttt
     5761 ctgtctgcag cttgtggcct gggtcacctc tacggctggc ccagatcctt ccctgccgcc
     5821 tccttcaggt tccgtcttcc tccactccct cttccccttg ctctctgctg tgttgctgcc
     5881 caaggatgct ctttccggag cacttccttc tcggcgctgc accacgtgat gtcctctgag
     5941 cggatcctcc ccgtgtctgg gtcctctccg ggcatctctc ctccctcacc caaccccatg
     6001 ccgtcttcac tcgctgggtt cccttttcct tctccttctg gggcctgtgc catctctcgt
     6061 ttcttaggat ggccttctcc gacggatgtc tcccttgcgt cccgcctccc cttcttgtag
     6121 gcctgcatca tcaccgtttt tctggacaac cccaaagtac cccgtctccc tggctttagc
     6181 cacctctcca tcctcttgct ttctttgcct ggacaccccg ttctcctgtg gattcgggtc
     6241 acctctcact cctttcattt gggcagctcc cctacccccc ttacctctct agtctgtgct
     6301 agctcttcca gccccctgtc atggcatctt ccaggggtcc gagagctcag ctagtcttct
     6361 tcctccaacc cgggccccta tgtccacttc aggacagcat gtttgctgcc tccagggatc
     6421 ctgtgtcccc gagctgggac caccttatat tcccagggcc ggttaatgtg gctctggttc
     6481 tgggtacttt tatctgtccc ctccacccca cagtggggca agcttattac cctgttatcc
     6541 ctaaccggtg cgtttttatg cttgtagtat tgtataatgt ttttaagatc caagcttctg
     6601 acctcttctc ttcctcccac agggcctcga gacaattgat taacatcgat acggtaccga
     6661 gttggcgcgc ctgggagctc acggggacag ccccccccca aagcccccag ggatgtaatt
     6721 acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg
     6781 ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc
     6841 acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg
     6901 gatacgggga aaaagcttta ggctgaaaga gagatttaga atgacaggcg cgcctggcca
     6961 tacatcgata cggtaccgag ttggcgcgcc tgggagctca cggggacagc ccccccccaa
     7021 agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg
     7081 gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc
     7141 cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga
     7201 gcctgcagac acctgggggg atacggggaa aaagctttag gctgaaagag agatttagaa
     7261 tgacaggcgc gcctggtggc gaacgat
//
