LOCUS       Exported                8012 bp ds-DNA     circular SYN 16-AUG-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pLPAR1_TCS(Q'L)_HA-dCas9(N)_P2A-Puro-WPRE+T285
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8012)
  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..8012
                     /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..1997
                     /codon_start=1
                     /label=LPAR1
                     /translation="MAAISTSIPVISQPQFTAMNEPQCFYNESIAFFYNRSGKHLATEW
                     NTVSKLVMGLGITVCIFIMLANLLVMVAIYVNRRFHFPIYYLMANLAAADFFAGLAYFY
                     LMFNTGPNTRRLTVSTWLLRQGLIDTSLTASVANLLAIAIERHITVFRMQLHTRMSNRR
                     VVVVIVVIWTMAIVMGAIPSVGWNCICDIENCSNMAPLYSDSYLVFWAIFNLVTFVVMV
                     VLYAHIFGYVRQRTMRMSRHSSGPRRNRDTMMSLLKTVVIVLGAFIICWTPGLVLLLLD
                     VCCPQCDVLAYEKFFLLLAEFNSAMNPIIYSYRDKEMSATFRQILCCQRSENPTGPTEG
                     SDRSASSLNHTILAGVHSNDHSVV"
     CDS             1998..2096
                     /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             2097..2117
                     /codon_start=1
                     /label=alternative TEV cleavage site
                     /translation="ENLYFQL"
     CDS             2124..2150
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2166..3767
                     /codon_start=1
                     /note="hSpCas9"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMR"
     misc_feature    3768..3785
                     /note="(GGGGS)3 linker"
     CDS             3788
                     /codon_start=1
                     /label=rtTA
                     /protein_id="rtTA-M2"
                     /translation=""
     CDS             3795..3854
                     /codon_start=1
                     /label=P2A
                     /translation="GATNFSLLKQAGDVEENPGP"
     CDS             3861..4454
                     /codon_start=1
                     /label=Puro
                     /protein_id="Puro"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKG"
     protein_bind    4491..5018
                     /label=protein bind
     misc_feature    5036..5267
                     /note="bGH polyA"
     misc_feature    5276..5416
                     /label=R-ITR
     rep_origin      5508..5814
                     /direction=RIGHT
                     /label=f1 Origin
     misc_feature    6333..7190
                     /label=AmpR
     rep_origin      7341..8008
                     /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 tcgatatggc cgctattagc accagtatcc cagttatctc tcagccccag tttacagcga
      961 tgaatgagcc tcagtgcttt tacaacgaaa gcatcgcctt tttctataac agatccggga
     1021 agcatctggc caccgaatgg aatacggtgt ctaaattggt catgggcctt ggtattaccg
     1081 tttgcatctt tatcatgctt gcaaacttgc tggtgatggt ggctatctac gtgaaccgca
     1141 gattccattt tccaatttac tacttgatgg caaatcttgc cgcagctgac ttcttcgcgg
     1201 ggttggcata tttctacttg atgtttaata ccgggcccaa cacccgcaga cttactgtct
     1261 caacttggct gttgcgccag ggactcatcg acacttccct gactgcctct gtggcaaacc
     1321 tgttggccat cgcaattgag agacacataa cggtgttccg aatgcaactt catacacgga
     1381 tgtcaaaccg gagggtggtg gtggtgatcg tggtgatctg gaccatggct atcgttatgg
     1441 gcgccattcc tagcgtgggt tggaattgca tttgcgacat cgagaactgt tccaatatgg
     1501 cacctctgta ttctgacagt tatctggttt tctgggccat ctttaatctc gtgacatttg
     1561 tcgtgatggt ggtactgtat gctcacatct tcggatacgt gagacaacgc acaatgcgga
     1621 tgtctcgaca cagcagcgga cccagacgga atcgggacac aatgatgtcc ctcctcaaaa
     1681 ccgtcgtaat cgtgttgggc gcatttatca tttgttggac ccccgggctc gtactgcttc
     1741 tgctcgacgt gtgttgtccc cagtgtgacg ttcttgccta tgagaagttc tttcttttgt
     1801 tggccgaatt caactccgca atgaatccta ttatttactc ttatcgagat aaagagatgt
     1861 ccgcaacttt tcggcagatc ctctgctgcc agaggagcga gaatcctaca ggccccactg
     1921 aaggttcaga tcggtcagct tcatccctca accacacaat cctggccggt gtacactcca
     1981 acgatcattc cgtggtcatc gataccggtg gacgcacccc acccagcctg ggtccccaag
     2041 atgagtcctg caccaccgcc agctcctccc tggccaagga cacttcatcg accggtgaga
     2101 acctgtactt ccagctaact agttacccat acgacgtacc agattacgct ggaggttctg
     2161 gatccgacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg
     2221 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
     2281 ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg
     2341 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
     2401 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
     2461 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
     2521 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
     2581 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
     2641 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     2701 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     2761 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     2821 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa
     2881 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     2941 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     3001 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     3061 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     3121 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     3181 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     3241 ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     3301 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     3361 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     3421 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     3481 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     3541 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     3601 aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata
     3661 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     3721 tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaggt ggcggtggct
     3781 cgggtaccgg atctggagcc accaattttt ctctgctgaa gcaggctggt gatgttgaag
     3841 aaaaccctgg acctgaattc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     3901 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     3961 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     4021 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     4081 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     4141 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     4201 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     4261 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     4321 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     4381 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     4441 cccgcaaggg gtaactaagt aaggatcgat ccaagatatc gtattcttaa ctatgttgct
     4501 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     4561 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     4621 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     4681 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     4741 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     4801 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
     4861 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     4921 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     4981 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc aattcctaga
     5041 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     5101 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     5161 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     5221 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa
     5281 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     5341 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     5401 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5461 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     5521 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5581 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5641 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5701 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5761 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5821 accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5881 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     5941 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     6001 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     6061 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     6121 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     6181 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     6241 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     6301 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6361 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6421 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6481 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     6541 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6601 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6661 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6721 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6781 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6841 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6901 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6961 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     7021 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     7081 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     7141 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7201 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7261 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7321 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     7381 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     7441 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     7501 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     7561 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     7621 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     7681 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     7741 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7801 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7861 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7921 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7981 tcctggcctt ttgctggcct tttgctcaca tg
//
