LOCUS       Exported                9015 bp ds-DNA     circular SYN 16-AUG-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pBDKRB2_TCS(Q'G)_NLS-HA-dCas9(C)T2A_DHFR_PCP_VP64+T301
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 9015)
  AUTHORS   Weatherall Institute
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 16, 2017 from SnapGene 3.3.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9015
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     misc_feature    12..810
                     /note="CBh"
     misc_feature    817..824
                     /label=konzak
     CDS             825..905
                     /codon_start=1
                     /product="p.363: At the 5′ end, we included a cleavable 
                     signal sequence to promote membrane localization23 and a 
                     Flag epitope tag to allow monitoring of cell-surface 
                     expression by immunohistochemistry.  -- Highlighted 11 mar 
                     2016
                     flag - DYKDDDDK but where is the ""K""?"
                     /label=membrane localisation signal+ FLAG
                     /translation="MKTIIALSYIFCLVFADYKDDDDASID"
     CDS             906..2078
                     /codon_start=1
                     /label=BDKBR2
                     /translation="MFSPWKISMFLSVREDSVPTTASFSADMLNVTLQGPTLNGTFAQS
                     KCPQVEWLGWLNTIQPPFLWVLFVLATLENIFVLSVFCLHKSSCTVAEIYLGNLAAADL
                     ILACGLPFWAITISNNFDWLFGETLCRVVNAIISMNLYSSICFLMLVSIDRYLALVKTM
                     SMGRMRGVRWAKLYSLVIWGCTLLLSSPMLVFRTMKEYSDEGHNVTACVISYPSLIWEV
                     FTNMLLNVVGFLLPLSVITFCTMQIMQVLRNNEMQKFKEIQTERRATVLVLVVLLLFII
                     CWLPFQISTFLDTLHRLGILSSCQDERIIDVITQIASFMAYSNSCLNPLVYVIVGKRFR
                     KKSWEVYQGVCQKGGCRSEPIQMENSMGTLRTSISVERQIHKLQDWAGSRQ"
     CDS             2079..2177
                     /codon_start=1
                     /label=crucial for arrestin recruitment
                     /note="added sequence from the C terminus of the V2 
                     vasopressin receptor (V2 tail) after each recep- tor 
                     sequence, to promote arrestin recruitment20,24–26.         
                                 "
                     /translation="IDTGGRTPPSLGPQDESCTTASSSLAKDTSSTG"
     CDS             2178..2198
                     /codon_start=1
                     /label=efficient TEV cleavage site
                     /translation="ENLYFQG"
     CDS             2199..2219
                     /codon_start=1
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2238..2264
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2265..2273
                     /label=GGS linker
     misc_feature    2280..2291
                     /note="(GGGGS)3 linker"
     CDS             2292..4790
                     /codon_start=1
                     /note="hSpCas9"
                     /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI
                     SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
                     AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH
                     DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE
                     NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL
                     QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEV
                     VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI
                     LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV
                     GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
                     ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL
                     PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME
                     RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP
                     SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD
                     KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL
                     IHQSITGLYETRIDLSQLGGD"
     misc_feature    4791..4823
                     /note="NLS"
     CDS             4836..4889
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4890..5363
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILER"
     CDS             5367..5411
                     /codon_start=1
                     /label=GGGGS linker
                     /translation="GGGGSGGGGSGGGGS"
     CDS             5412..5774
                     /codon_start=1
                     /label=PP7
                     /translation="SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLR
                     QNGAKTAYRVNLKLDQADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLV
                     ATSQVEDLVVNLVPLGR"
     CDS             5781..5825
                     /codon_start=1
                     /label=GGGGS- linker
                     /translation="GGGGSGGGGSGGGGS"
     CDS             5829..5849
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5874..6023
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     misc_feature    6039..6270
                     /note="bGH polyA"
     misc_feature    6279..6419
                     /label=R-ITR
     rep_origin      6511..6817
                     /direction=RIGHT
                     /label=f1 Origin
     misc_feature    7336..8193
                     /label=AmpR
     rep_origin      8344..9011
                     /direction=RIGHT
                     /label=pUC ori
ORIGIN
        1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg
      601 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      661 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa
      721 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca
      781 cctgcctgaa atcacttttt ttcaggttgg accggtgcgc caccatgaag acgatcatcg
      841 ccctgagcta catcttctgc ctggtattcg ccgactacaa ggacgatgat gacgccagca
      901 tcgatatgtt ctcaccctgg aagatttcta tgttcctttc tgtgcgcgag gacagcgtgc
      961 ccacaactgc gagttttagc gccgacatgc ttaacgtgac cctgcagggc cccactctca
     1021 acgggacatt tgctcagtct aaatgtcctc aagtggaatg gctcggctgg ttgaacacta
     1081 tccagccccc tttcctttgg gttctttttg ttctggccac gctggaaaac atctttgttc
     1141 tctctgtgtt ttgcctccat aagagctcct gtacggtggc cgaaatttac ctgggtaatt
     1201 tggccgcagc ggatctcatt ttggcctgtg gcctgccttt ctgggctatt actatcagca
     1261 ataattttga ttggttgttt ggcgagacct tgtgtcgcgt ggtgaatgca attatcagta
     1321 tgaatctgta cagctcaatc tgcttcctta tgctcgtcag cattgacagg tacctcgccc
     1381 ttgtaaagac tatgagcatg ggccgcatga ggggcgtacg ctgggctaag ctctatagcc
     1441 tggtgatttg gggttgcacc ctcctcctca gttcacctat gctggtcttc cgaacgatga
     1501 aggagtactc tgacgagggc cacaatgtta ccgcatgcgt tatcagttac ccatctttga
     1561 tttgggaggt cttcacaaac atgctgctga acgtggtggg ttttttgctc cccctttctg
     1621 tgattacctt ctgtacaatg cagatcatgc aagttttgcg aaacaacgaa atgcaaaaat
     1681 ttaaggagat acagaccgag cgaagggcca cagtgctggt cttggtcgtt ttgctgttgt
     1741 tcattatctg ttggctgcct ttccagatca gcactttcct ggacacgttg cacagactcg
     1801 gaatcctgag tagttgtcag gacgaaagga tcatcgacgt tatcactcaa atcgcgtcct
     1861 tcatggcata ctccaatagc tgcttgaacc cattggtgta tgttattgtg ggaaaacggt
     1921 ttaggaagaa gagctgggaa gtgtaccaag gggtgtgcca aaagggaggc tgccgatcag
     1981 agccaatcca aatggaaaat agcatgggta ccctgagaac ctccattagc gtggagcgac
     2041 agatccacaa gctccaggac tgggctggct ctagacagat cgataccggt ggacgcaccc
     2101 cacccagcct gggtccccaa gatgagtcct gcaccaccgc cagctcctcc ctggccaagg
     2161 acacttcatc gaccggtgag aacctgtact tccagggccc caagaagaag aggaaggtgg
     2221 ggggaggcag cactagttac ccatacgacg taccagatta cgctggaggt tctggatccg
     2281 gcggtggctc gaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     2341 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     2401 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     2461 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     2521 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     2581 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     2641 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     2701 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     2761 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     2821 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     2881 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     2941 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     3001 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     3061 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     3121 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     3181 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     3241 tcgacaacaa ggtgctgacc agaagcgaca aggcccgggg caagagcgac aacgtgccct
     3301 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     3361 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     3421 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     3481 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     3541 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     3601 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     3661 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     3721 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     3781 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     3841 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     3901 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     3961 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     4021 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     4081 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     4141 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     4201 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     4261 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     4321 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     4381 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     4441 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     4501 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     4561 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     4621 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     4681 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     4741 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac agccccaaga
     4801 agaagagaaa ggtggaggcc agcagcggca gtggagaggg cagaggaagt ctgctaacat
     4861 gcggtgacgt cgaggagaat cctggcccaa tgatcagtct gattgcggcg ttagcggtag
     4921 attacgttat cggcatggaa aacgccatgc cgtggaacct gcctgccgat ctcgcctggt
     4981 ttaaacgcaa caccttaaat aaacccgtga ttatgggccg ccatacctgg gaatcaatcg
     5041 gtcgtccgtt gccaggacgc aaaaatatta tcctcagcag tcaaccgagt acggacgatc
     5101 gcgtaacgtg ggtgaagtcg gtggatgaag ccatcgcggc gtgtggtgac gtaccagaaa
     5161 tcatggtgat tggcggcggt cgcgttattg aacagttctt gccaaaagcg caaaaactgt
     5221 atctgacgca tatcgacgca gaagtggaag gcgacaccca tttcccggat tacgagccgg
     5281 atgactggga atcggtattc agcgaattcc acgatgctga tgcgcagaac tctcacagct
     5341 attgctttga gattctggag cggcgaggtg gaggaggttc aggaggtgga ggctctggag
     5401 gtggaggcag ctccaaaacc atcgttcttt cggtcggcga ggctactcgc actctgactg
     5461 agatccagtc caccgcagac cgtcagatct tcgaagagaa ggtcgggcct ctggtgggtc
     5521 ggctgcgcct cacggcttcg ctccgtcaaa acggagccaa gaccgcgtat cgcgtcaacc
     5581 taaaactgga tcaggcggac gtcgttgatt ccggacttcc gaaagtgcgc tacactcagg
     5641 tatggtcgca cgacgtgaca atcgttgcga atagcaccga ggcctcgcgc aaatcgttgt
     5701 acgatttgac caagtccctc gtcgcgacct cgcaggtcga agatcttgtc gtcaaccttg
     5761 tgccgctggg ccgtagcgct ggaggaggtg gaagcggagg aggaggaagc ggaggaggag
     5821 gtagcggacc taagaaaaag aggaaggtgg cggccgctgg atccggacgg gctgacgcat
     5881 tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt gaccttgaca
     5941 tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt gacgcccttg
     6001 atgatttcga cctggacatg ctgattaact aagaattcct agagctcgct gatcagcctc
     6061 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     6121 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     6181 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     6241 ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt
     6301 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     6361 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     6421 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     6481 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     6541 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     6601 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     6661 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     6721 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     6781 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     6841 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     6901 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     6961 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     7021 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     7081 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     7141 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     7201 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     7261 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     7321 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     7381 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     7441 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     7501 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     7561 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     7621 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     7681 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     7741 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     7801 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     7861 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     7921 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     7981 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     8041 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     8101 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     8161 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     8221 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     8281 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     8341 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     8401 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     8461 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     8521 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     8581 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     8641 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     8701 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     8761 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     8821 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     8881 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     8941 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     9001 ccttttgctc acatg
//
