LOCUS       eSpCas9(1.1)_No_        8439 bp ds-DNA     circular SYN 28-JUN-2021
DEFINITION  Expresses the AAVS1 T2 gRNA in combination with FLAGless
            eSpCas9(1.1) to target the AAVS1 ""safe harbor"" locus.
ACCESSION
VERSION     .
KEYWORDS    eSpCas9(1.1)_No_FLAG_AAVS1_T2.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 8439)
  AUTHORS   Dalvai M, Loehr J, Jacquet K, Huard CC, Roques C, Herst P, Cote J
            and Doyon Y
  TITLE     A Scalable Genome-Editing-Based Approach for Mapping Multiprotein
            Complexes in Human Cells.
  JOURNAL   Cell Rep. 2015 Oct 7. pii: S2211-1247(15)01020-7. doi:
            10.1016/j.celrep.2015.09.009.
  PUBMED    26456817
REFERENCE   2  (bases 1 to 8439)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     promoter        1..241
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     primer_bind     1..21
                     /note="Human U6 promoter, forward primer"
                     /standard_name="hU6-F"
     primer_bind     172..191
                     /note="Human U6 promoter, forward primer"
                     /standard_name="LKO.1 5'"
     misc_RNA        250..269
                     /standard_name="ATP1A1 G7"
     misc_RNA        270..345
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /standard_name="sgRNA scaffold"
     enhancer        442..727
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV
                     enhancer"
                     /standard_name="CMV enhancer"
     promoter        729..1006
                     /standard_name="chicken beta-actin promoter"
     intron          1007..1235
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
                     /standard_name="hybrid intron"
     regulatory      1247..1256
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation
                      of translation (Kozak, 1987)"
     CDS             1259..1279
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian
                     virus 40) large T antigen"
                     /translation="PKKKRKV"
                     /standard_name="SV40 NLS"
     CDS             1304..5404
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system, mutated to improve
                     targeting specificity (Slaymaker et al., 2016)"
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE
                     SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM
                     IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKS
                     RRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
                     LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL
                     KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL
                     NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY
                     VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP
                     KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED
                     YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTL
                     FEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL
                     KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTV
                     KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH
                     PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVL
                     TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG
                     FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY
                     KVREINNYHHAHDAYLNAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSM
                     PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA
                     KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE
                     LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHK
                     HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP
                     AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /standard_name="eSpCas9(1.1)"
     CDS             5405..5452
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
                     /standard_name="nucleoplasmin NLS"
     primer_bind     complement(5480..5497)
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /standard_name="BGH-rev"
     polyA_signal    5486..5693
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5702..5842
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /standard_name="AAV2 ITR"
     repeat_region   5702..5831
                     /standard_name="AAV2 ITR"
     rep_origin      5917..6372
     primer_bind     complement(6004..6023)
                     /note="F1 origin, reverse primer"
                     /standard_name="F1ori-R"
     primer_bind     6214..6235
                     /note="F1 origin, forward primer"
                     /standard_name="F1ori-F"
     primer_bind     complement(6389..6408)
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /standard_name="pRS-marker"
     primer_bind     6508..6530
                     /note="pGEX vectors, reverse primer"
                     /standard_name="pGEX 3'"
     primer_bind     complement(6568..6586)
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
                     /standard_name="pBRforEco"
     promoter        6654..6758
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             6759..7619
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /standard_name="AmpR"
     primer_bind     complement(6977..6996)
                     /note="Ampicillin resistance gene, reverse primer"
                     /standard_name="Amp-R"
     rep_origin      7790..8378
     primer_bind     8279..8298
                     /note="pBR322 origin, forward primer"
                     /standard_name="pBR322ori-F"
ORIGIN      
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg tcacagatcg atagtagtgg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
      481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
      541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
      661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      841 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
      901 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac
     1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
     1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggcccc
     1261 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     1321 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     1381 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     1441 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     1501 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     1561 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     1621 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     1681 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     1741 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     1801 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     1861 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     1921 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     1981 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     2041 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     2101 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     2161 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     2221 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     2281 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     2341 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     2401 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     2461 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     2521 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     2581 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     2641 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     2701 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     2761 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     2821 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     2881 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     2941 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     3001 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     3061 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     3121 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     3181 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     3241 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     3301 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     3361 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     3421 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     3481 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     3541 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     3601 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     3661 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     3721 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     3781 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     3841 ggccgacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     3901 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     3961 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     4021 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     4081 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     4141 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     4201 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     4261 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctgcgc tggaaagcga
     4321 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     4381 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     4441 gaccgagatt accctggcca acggcgagat ccggaaggcg cctctgatcg agacaaacgg
     4501 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     4561 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     4621 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     4681 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     4741 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     4801 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     4861 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     4921 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     4981 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     5041 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     5101 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     5161 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     5221 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     5281 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     5341 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     5401 cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt
     5461 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     5521 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     5581 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     5641 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     5701 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5761 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5821 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5881 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5941 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     6001 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     6061 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     6121 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     6181 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     6241 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     6301 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     6361 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6421 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6481 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6541 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     6601 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6661 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6721 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6781 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6841 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6901 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6961 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     7021 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     7081 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     7141 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     7201 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     7261 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     7321 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7381 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7441 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     7501 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7561 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7621 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7681 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7741 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7801 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7861 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7921 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7981 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     8041 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     8101 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     8161 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     8221 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     8281 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     8341 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8401 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
