LOCUS       eSpCas9(1.1)_No_        8440 bp ds-DNA     circular SYN 19-OCT-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..270
                     /standard_name="LMNA G2"
     misc_RNA        271..346
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /standard_name="sgRNA scaffold"
     terminator      347..352
                     /standard_name="U6 terminator"
     enhancer        443..728
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV
                     enhancer"
                     /standard_name="CMV enhancer"
     promoter        730..1007
                     /standard_name="chicken beta-actin promoter"
     intron          1008..1236
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
                     /standard_name="hybrid intron"
     regulatory      1248..1257
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation
                      of translation (Kozak, 1987)"
     CDS             1260..1280
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian
                     virus 40) large T antigen"
                     /translation="PKKKRKV"
                     /standard_name="SV40 NLS"
     CDS             1305..5405
                     /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             5406..5453
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
                     /standard_name="nucleoplasmin NLS"
     primer_bind     complement(5481..5498)
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /standard_name="BGH-rev"
     polyA_signal    5487..5694
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5703..5843
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /standard_name="AAV2 ITR"
     repeat_region   5703..5832
                     /standard_name="AAV2 ITR"
     rep_origin      5918..6373
     primer_bind     complement(6005..6024)
                     /note="F1 origin, reverse primer"
                     /standard_name="F1ori-R"
     primer_bind     6215..6236
                     /note="F1 origin, forward primer"
                     /standard_name="F1ori-F"
     primer_bind     complement(6390..6409)
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /standard_name="pRS-marker"
     primer_bind     6509..6531
                     /note="pGEX vectors, reverse primer"
                     /standard_name="pGEX 3'"
     primer_bind     complement(6569..6587)
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
                     /standard_name="pBRforEco"
     promoter        6655..6759
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             6760..7620
                     /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(6978..6997)
                     /note="Ampicillin resistance gene, reverse primer"
                     /standard_name="Amp-R"
     rep_origin      7791..8379
     primer_bind     8280..8299
                     /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 ccatggagac cccgtcccag gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga
     1021 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1081 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1141 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat
     1201 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggccc
     1261 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     1321 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     1381 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     1441 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     1501 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     1561 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     1621 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     1681 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     1741 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     1801 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     1861 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     1921 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     1981 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     2041 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     2101 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     2161 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     2221 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     2281 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     2341 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     2401 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     2461 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     2521 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     2581 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     2641 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     2701 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     2761 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     2821 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     2881 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     2941 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     3001 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     3061 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     3121 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     3181 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     3241 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     3301 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     3361 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     3421 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     3481 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     3541 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     3601 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     3661 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     3721 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     3781 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     3841 tggccgacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     3901 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     3961 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     4021 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     4081 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     4141 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     4201 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     4261 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctgcg ctggaaagcg
     4321 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     4381 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     4441 agaccgagat taccctggcc aacggcgaga tccggaaggc gcctctgatc gagacaaacg
     4501 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     4561 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     4621 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     4681 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     4741 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     4801 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     4861 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     4921 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     4981 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     5041 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     5101 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     5161 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     5221 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     5281 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     5341 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     5401 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaagaat
     5461 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     5521 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     5581 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     5641 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     5701 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5761 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5821 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5881 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5941 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     6001 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     6061 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     6121 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     6181 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     6241 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     6301 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     6361 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6421 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6481 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6541 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6601 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6661 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6721 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6781 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6841 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6901 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6961 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     7021 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     7081 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     7141 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     7201 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     7261 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     7321 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7381 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7441 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     7501 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     7561 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7621 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7681 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7741 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7801 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7861 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7921 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     7981 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     8041 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     8101 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     8161 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     8221 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     8281 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     8341 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     8401 ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
