LOCUS       Exported                8033 bp ds-DNA     circular SYN 16-AUG-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pAVPR2_TCS(Q'L)_HA-dCas9(N)_P2A-Puro-WPRE+T198
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8033)
  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..8033
                     /organism="synthetic DNA construct"
                     /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..2018
                     /codon_start=1
                     /label=>AVPR2
                     /translation="MLMASTTSAVPGHPSLPSLPSNSSQERPLDTRDPLLARAELALLS
                     IVFVAVALSNGLVLAALARRGRRGHWAPIHVFIGHLCLADLAVALFQVLPQLAWKATDR
                     FRGPDALCRAVKYLQMVGMYASSYMILAMTLDRHRAICRPMLAYRHGSGAHWNRPVLVA
                     WAFSLLLSLPQLFIFAQRNVEGGSGVTDCWACFAEPWGRRTYVTWIALMVFVAPTLGIA
                     ACQVLIFREIHASLVPGPSERPGGRRRGRRTGSPGEGAHVSAAVAKTVRMTLVIVVVYV
                     LCWAPFFLVQLWAAWDPEAPLEGAPFVLLMLLASLNSCTNPWIYASFSSSVSSELRSLL
                     CCARGRTPPSLGPQDESCTTASSSLAKDTSS"
     CDS             2019..2117
                     /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             2118..2138
                     /codon_start=1
                     /label=alternative TEV cleavage site
                     /translation="ENLYFQL"
     CDS             2145..2171
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2187..3788
                     /codon_start=1
                     /note="hSpCas9"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMR"
     misc_feature    3789..3806
                     /note="(GGGGS)3 linker"
     CDS             3816..3875
                     /codon_start=1
                     /label=P2A
                     /translation="GATNFSLLKQAGDVEENPGP"
     CDS             3882..4475
                     /codon_start=1
                     /label=Puro
                     /protein_id="Puro"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKG"
     protein_bind    4512..5039
                     /label=protein bind
     misc_feature    5057..5288
                     /note="bGH polyA"
     misc_feature    5297..5437
                     /label=R-ITR
     rep_origin      5529..5835
                     /direction=RIGHT
                     /label=f1 Origin
     misc_feature    6354..7211
                     /label=AmpR
     rep_origin      7362..8029
                     /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 tcgatatgct catggcctct accacaagcg ctgtgcccgg acatccatct cttccaagtc
      961 ttccctccaa cagctcacag gagaggcctc tggatacccg ggacccactt cttgcaagag
     1021 cggagctcgc cttgctctcc atcgttttcg ttgccgtcgc gttgagcaat ggtctggtgc
     1081 tggccgccct ggcgcgacga ggccgccgag gccactgggc gcctattcat gtctttatcg
     1141 gccatctctg tcttgcagac ctggccgtgg cgctgtttca ggtgctccct caactggctt
     1201 ggaaagccac cgatagattc cgggggccag acgcactgtg tcgggccgtg aaatacctgc
     1261 agatggtggg aatgtatgcc agttcatata tgatacttgc tatgaccttg gacagacaca
     1321 gggctatctg tcgccccatg ctggcgtata ggcacggttc cggagcccat tggaacaggc
     1381 cagttctggt tgcgtgggct ttttccctgc ttctgagctt gccccagttg tttattttcg
     1441 cacagagaaa tgtggagggc ggatctggcg tgaccgattg ctgggcttgc ttcgctgagc
     1501 catggggcag aaggacctac gtgacgtgga ttgctctcat ggtgttcgtt gctcccaccc
     1561 tggggatcgc cgcatgccag gtcctgatat tcagagaaat acacgcctcc ttggttcctg
     1621 ggccaagcga gcgccctggt ggaagaagac ggggtcgaag gactggctct ccaggcgaag
     1681 gcgcccacgt atcagctgcg gtagctaaga ccgtacgcat gacactggtc atcgtcgtcg
     1741 tttacgttct ttgctgggcc ccgttttttt tggtgcagct ctgggcggcc tgggaccccg
     1801 aagcacccct ggaaggtgct ccattcgttc tgctcatgct tctcgcttca ctgaatagct
     1861 gtaccaatcc ttggatttac gcctctttct cctccagtgt tagctccgag ctgcggagtc
     1921 ttctctgttg cgcccggggc aggacccccc caagcctggg tccacaggac gaatcctgca
     1981 ctaccgcctc cagttccctg gccaaagaca cctcttctat cgataccggt ggacgcaccc
     2041 cacccagcct gggtccccaa gatgagtcct gcaccaccgc cagctcctcc ctggccaagg
     2101 acacttcatc gaccggtgag aacctgtact tccagctaac tagttaccca tacgacgtac
     2161 cagattacgc tggaggttct ggatccgaca agaagtacag catcggcctg gccatcggca
     2221 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     2281 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     2341 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     2401 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
     2461 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
     2521 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
     2581 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
     2641 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
     2701 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     2761 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
     2821 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
     2881 agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca
     2941 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     3001 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     3061 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     3121 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     3181 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     3241 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     3301 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     3361 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     3421 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     3481 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     3541 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     3601 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     3661 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     3721 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     3781 gaatgagagg tggcggtggc tcgggtaccg gatctggagc caccaatttt tctctgctga
     3841 agcaggctgg tgatgttgaa gaaaaccctg gacctgaatt catgaccgag tacaagccca
     3901 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     3961 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     4021 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     4081 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     4141 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     4201 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     4261 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     4321 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     4381 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     4441 gaccgcgcac ctggtgcatg acccgcaagg ggtaactaag taaggatcga tccaagatat
     4501 cgtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4561 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4621 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4681 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4741 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     4801 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     4861 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     4921 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     4981 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5041 gcctccccgc caattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     5101 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5161 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5221 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     5281 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     5341 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     5401 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     5461 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     5521 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5581 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5641 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5701 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5761 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5821 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     5881 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5941 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     6001 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     6061 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     6121 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     6181 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     6241 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6301 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6361 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6421 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6481 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6541 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6601 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6661 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6721 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6781 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6841 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6901 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6961 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     7021 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     7081 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     7141 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7201 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7261 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7321 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7381 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7441 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7501 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     7561 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7621 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7681 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7741 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7801 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7861 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7921 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7981 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atg
//
