LOCUS       40924_42184        7299 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT20_T20_72bp-mKate2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7299)
  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  JOURNAL   Unpublished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 7299)
  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 7299)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7299)
  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            "Unpublished"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..7299
                     /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..201
                     /label=TALER20 bs binding site
                     /bound_moiety="TALER20 bs"
     misc_feature    208..267
                     /label=CMVmini
                     /note="CMVmini"
     protein_bind    274..298
                     /label=TALER20 bs binding site
                     /bound_moiety="TALER20 bs"
     protein_bind    315..335
                     /label=attB1
                     /note="core recombination site for the Gateway(R) BP
                     reaction"
     CDS             377..394
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             395..1090
                     /codon_start=1
                     /label=mKate2
                     /note="monomeric far-red fluorescent protein (Shcherbo et
                     al., 2009)"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHK"
     protein_bind    complement(1133..1157)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1312..1367
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1728..1744)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1752..1768
                     /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(1776..1806)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1821..1842
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    2015..2851
                     /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(2881..2899)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2920..2936)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2944..2960)
                     /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(2968..2998)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3013..3034)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3322..3910)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4084..4941)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4942..5046)
                     /label=AmpR promoter
     rep_origin      complement(5072..5527)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     5669..5685
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5695..5713
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    5728..6531
                     /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    6610..6635
                     /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 tctagatcta
      181 gactcttcac aatgttctta tgaattctag gcgtgtacgg tgggaggcct atataagcag
      241 agctcgttta gtgaaccgtc agatcgcctc gagtctagac tcttcacaat gttcttatgg
      301 atccaattcg accccaagtt tgtacaaaaa agcagggatc ccggtaccct cgacacctgc
      361 tgacggagcc accatgcacc atcaccatca ccatatggtg agcgagctga ttaaggagaa
      421 catgcacatg aagctgtaca tggagggcac cgtgaacaac caccacttca agtgcacatc
      481 cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg cggtcgaggg
      541 cggccctctc cccttcgcct tcgacatcct ggctaccagc ttcatgtacg gcagcaaaac
      601 cttcatcaac cacacccagg gcatccccga cttctttaag cagtccttcc ccgagggctt
      661 cacatgggag agagtcacca catacgaaga tgggggcgtg ctgaccgcta cccaggacac
      721 cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcccatc
      781 caacggccct gtgatgcaga agaaaacact cggctgggag gcctccaccg agacactgta
      841 ccccgctgac ggcggcctgg aaggcagagc cgacatggcc ctgaagctcg tgggcggggg
      901 ccacctgatc tgcaacctta agaccacata cagatccaag aaacccgcta agaacctcaa
      961 gatgcccggc gtctactatg tggacaggag actggaaaga atcaaggagg ccgacaaaga
     1021 gacatacgtc gagcagcacg aggtggctgt ggccagatac tgcgacctcc ctagcaaact
     1081 ggggcacaaa cttaattcct agtaacgctc agtgcaggtg aattctacca ccacccagct
     1141 ttcttgtaca aagtggtacg cgtgaattca ctcctcaggt gcaggctgcc tatcagaagg
     1201 tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg
     1261 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga
     1321 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
     1381 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
     1441 atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga
     1501 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt
     1561 tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg
     1621 ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc
     1681 tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt
     1741 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     1801 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1861 cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcg acagtactaa gcttggtcta
     1921 atacccaatc tctcgtctta tccagatgtt ttatacgcct taattaatag ggataacagg
     1981 gtaatctcga ggtttaaacg tcgacgcggc cgctactagg gacaggattg gtgacagaaa
     2041 agccccatcc ttaggcctcc tccttcctag tctcctgata ttgggtctaa cccccacctc
     2101 ctgttaggca gattccttat ctggtgacac acccccattt cctggagcca tctctctcct
     2161 tgccagaacc tctaaggttt gcttacgatg gagccagaga ggatcctggg agggagagct
     2221 tggcaggggg tgggagggaa gggggggatg cgtgacctgc ccggttctca gtggccaccc
     2281 tgcgctaccc tctcccagaa cctgagctgc tctgacgcgg ctgtctggtg cgtttcactg
     2341 atcctggtgc tgcagcttcc ttacacttcc caagaggaga agcagtttgg aaaaacaaaa
     2401 tcagaataag ttggtcctga gttctaactt tggctcttca cctttctagt ccccaattta
     2461 tattgttcct ccgtgcgtca gttttacctg tgagataagg ccagtagcca gccccgtcct
     2521 ggcagggctg tggtgaggag gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg
     2581 cgtcctaggt gttcaccagg tcgtggccgc ctctactccc tttctctttc tccatccttc
     2641 tttccttaaa gagtccccag tgctatctgg gacatattcc tccgcccaga gcagggtccc
     2701 gcttccctaa ggccctgctc tgggcttctg ggtttgagtc cttggcaagc ccaggagagg
     2761 cgctcaggct tccctgtccc ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc
     2821 ccttccctac aggggttcct ggctctgctc tccaccgcgg tggagctcca gcttttgttc
     2881 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     2941 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3001 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     3061 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3121 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3181 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3241 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3301 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3361 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3421 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3481 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3541 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3601 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     3661 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     3721 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3781 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3841 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3901 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3961 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4021 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4081 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4141 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4201 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4261 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4321 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4381 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4441 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     4501 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     4561 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     4621 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     4681 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     4741 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     4801 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4861 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     4921 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4981 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5041 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     5101 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5161 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5221 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5281 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5341 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     5401 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     5461 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     5521 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     5581 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     5641 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
     5701 gactcactat agggcgaatt gggtacctgc tttctctgac cagcattctc tcccctgggc
     5761 ctgtgccgct ttctgtctgc agcttgtggc ctgggtcacc tctacggctg gcccagatcc
     5821 ttccctgccg cctccttcag gttccgtctt cctccactcc ctcttcccct tgctctctgc
     5881 tgtgttgctg cccaaggatg ctctttccgg agcacttcct tctcggcgct gcaccacgtg
     5941 atgtcctctg agcggatcct ccccgtgtct gggtcctctc cgggcatctc tcctccctca
     6001 cccaacccca tgccgtcttc actcgctggg ttcccttttc cttctccttc tggggcctgt
     6061 gccatctctc gtttcttagg atggccttct ccgacggatg tctcccttgc gtcccgcctc
     6121 cccttcttgt aggcctgcat catcaccgtt tttctggaca accccaaagt accccgtctc
     6181 cctggcttta gccacctctc catcctcttg ctttctttgc ctggacaccc cgttctcctg
     6241 tggattcggg tcacctctca ctcctttcat ttgggcagct cccctacccc ccttacctct
     6301 ctagtctgtg ctagctcttc cagccccctg tcatggcatc ttccaggggt ccgagagctc
     6361 agctagtctt cttcctccaa cccgggcccc tatgtccact tcaggacagc atgtttgctg
     6421 cctccaggga tcctgtgtcc ccgagctggg accaccttat attcccaggg ccggttaatg
     6481 tggctctggt tctgggtact tttatctgtc ccctccaccc cacagtgggg caagcttatt
     6541 accctgttat ccctaaccgg tgcgttttta tgcttgtagt attgtataat gtttttaaga
     6601 tccaagcttc tgacctcttc tcttcctccc acagggcctc gagacaattg attaacatcg
     6661 atacggtacc gagttggcgc gcctgggagc tcacggggac agcccccccc caaagccccc
     6721 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc
     6781 tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac
     6841 ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca
     6901 gacacctggg gggatacggg gaaaaagctt taggctgaaa gagagattta gaatgacagg
     6961 cgcgcctggc catacatcga tacggtaccg agttggcgcg cctgggagct cacggggaca
     7021 gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc
     7081 gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt
     7141 gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc
     7201 gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt aggctgaaag
     7261 agagatttag aatgacaggc gcgcctggtg gcgaacgat
//
