LOCUS       40924_42189        7325 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT21_T21x3_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7325)
  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  JOURNAL   Unpublished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 7325)
  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 7325)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7325)
  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            "Unpublished"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..7325
                     /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=TALER21 bs binding site
                     /bound_moiety="TALER21 bs"
     misc_feature    202..261
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    268..286
                     /label=TALER21 bs binding site
                     /bound_moiety="TALER21 bs"
     protein_bind    287..305
                     /label=TALER21 bs binding site
                     /bound_moiety="TALER21 bs"
     protein_bind    306..324
                     /label=TALER21 bs binding site
                     /bound_moiety="TALER21 bs"
     protein_bind    341..361
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             403..420
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             421..1116
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1159..1183)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1338..1393
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1754..1770)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1778..1794
                     /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(1802..1832)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1847..1868
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2041..2877
                     /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(2907..2925)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2946..2962)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2970..2986)
                     /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(2994..3024)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3039..3060)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3348..3936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4110..4967)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4968..5072)
                     /label=AmpR promoter
     rep_origin      complement(5098..5553)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5695..5711
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5721..5739
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5754..6557
                     /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    6636..6661
                     /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 tctagatgtc
      181 gtcttctgca cttatgaatt ctaggcgtgt acggtgggag gcctatataa gcagagctcg
      241 tttagtgaac cgtcagatcg cctcgagtgt cgtcttctgc acttattgtc gtcttctgca
      301 cttattgtcg tcttctgcac ttatggatcc aattcgaccc caagtttgta caaaaaagca
      361 gggatcccgg taccctcgac acctgctgac ggagccacca tgcaccatca ccatcaccat
      421 atggtgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
      481 aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
      541 accatgagaa tcaaggcggt cgagggcggc cctctcccct tcgccttcga catcctggct
      601 accagcttca tgtacggcag caaaaccttc atcaaccaca cccagggcat ccccgacttc
      661 tttaagcagt ccttccccga gggcttcaca tgggagagag tcaccacata cgaagatggg
      721 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
      781 aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc
      841 tgggaggcct ccaccgagac actgtacccc gctgacggcg gcctggaagg cagagccgac
      901 atggccctga agctcgtggg cgggggccac ctgatctgca accttaagac cacatacaga
      961 tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga caggagactg
     1021 gaaagaatca aggaggccga caaagagaca tacgtcgagc agcacgaggt ggctgtggcc
     1081 agatactgcg acctccctag caaactgggg cacaaactta attcctagta acgctcagtg
     1141 caggtgaatt ctaccaccac ccagctttct tgtacaaagt ggtacgcgtg aattcactcc
     1201 tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa
     1261 ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     1321 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     1381 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     1441 gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg
     1501 gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag
     1561 aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt
     1621 aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact
     1681 actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct
     1741 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     1801 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1861 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1921 ggatcgacag tactaagctt ggtctaatac ccaatctctc gtcttatcca gatgttttat
     1981 acgccttaat taatagggat aacagggtaa tctcgaggtt taaacgtcga cgcggccgct
     2041 actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc
     2101 ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc
     2161 ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc
     2221 cagagaggat cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg
     2281 acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg
     2341 acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag
     2401 aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc
     2461 tcttcacctt tctagtcccc aatttatatt gttcctccgt gcgtcagttt tacctgtgag
     2521 ataaggccag tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt
     2581 ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct
     2641 actccctttc tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca
     2701 tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt
     2761 tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac
     2821 catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctcca
     2881 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     2941 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3001 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3061 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3121 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3181 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3241 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3301 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3361 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3421 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3481 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3541 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3601 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3661 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3721 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3781 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3841 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3901 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3961 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4021 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4081 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4141 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4201 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4261 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4321 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4381 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4441 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4501 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4561 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4621 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4681 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4741 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4801 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4861 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4921 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4981 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5041 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     5101 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5161 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5221 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5281 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     5341 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     5401 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     5461 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     5521 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     5581 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     5641 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     5701 cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt acctgctttc
     5761 tctgaccagc attctctccc ctgggcctgt gccgctttct gtctgcagct tgtggcctgg
     5821 gtcacctcta cggctggccc agatccttcc ctgccgcctc cttcaggttc cgtcttcctc
     5881 cactccctct tccccttgct ctctgctgtg ttgctgccca aggatgctct ttccggagca
     5941 cttccttctc ggcgctgcac cacgtgatgt cctctgagcg gatcctcccc gtgtctgggt
     6001 cctctccggg catctctcct ccctcaccca accccatgcc gtcttcactc gctgggttcc
     6061 cttttccttc tccttctggg gcctgtgcca tctctcgttt cttaggatgg ccttctccga
     6121 cggatgtctc ccttgcgtcc cgcctcccct tcttgtaggc ctgcatcatc accgtttttc
     6181 tggacaaccc caaagtaccc cgtctccctg gctttagcca cctctccatc ctcttgcttt
     6241 ctttgcctgg acaccccgtt ctcctgtgga ttcgggtcac ctctcactcc tttcatttgg
     6301 gcagctcccc tacccccctt acctctctag tctgtgctag ctcttccagc cccctgtcat
     6361 ggcatcttcc aggggtccga gagctcagct agtcttcttc ctccaacccg ggcccctatg
     6421 tccacttcag gacagcatgt ttgctgcctc cagggatcct gtgtccccga gctgggacca
     6481 ccttatattc ccagggccgg ttaatgtggc tctggttctg ggtactttta tctgtcccct
     6541 ccaccccaca gtggggcaag cttattaccc tgttatccct aaccggtgcg tttttatgct
     6601 tgtagtattg tataatgttt ttaagatcca agcttctgac ctcttctctt cctcccacag
     6661 ggcctcgaga caattgatta acatcgatac ggtaccgagt tggcgcgcct gggagctcac
     6721 ggggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc cgctaggggg
     6781 cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat ccccgagccg
     6841 gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt tcctctgaac
     6901 gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa aagctttagg
     6961 ctgaaagaga gatttagaat gacaggcgcg cctggccata catcgatacg gtaccgagtt
     7021 ggcgcgcctg ggagctcacg gggacagccc ccccccaaag cccccaggga tgtaattacg
     7081 tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc
     7141 cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg
     7201 ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat
     7261 acggggaaaa agctttaggc tgaaagagag atttagaatg acaggcgcgc ctggtggcga
     7321 acgat
//
