LOCUS       pAAV-R26-FLEX-mStr__        7213 bp ds-DNA     circular SYN 11-NOV-2019
DEFINITION  A single vector AAV-Cas9 system containing Cas9 from Staphylococcus
            aureus (SaCas9) and its sgRNA.
ACCESSION
VERSION     .
KEYWORDS    pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA; U6::BsaI-sgRNA.
SOURCE      
  ORGANISM  .
REFERENCE   1  (bases 1 to 7446)
  AUTHORS   Ran, FA, Cong, L, Yan, WX, Scott, DA, Gootenberg, JS, Kriz, AJ,
            Zetsche, B, Shalem, O, Wu, X, Makarova, KS, Koonin, EV, Sharp, PA,
            Zhang and F
  TITLE     In vivo genome editing using Staphylococcus aureus Cas9.
  JOURNAL   Nature. 2015 Apr 1. doi: 10.1038/nature14299.
  PUBMED    25830891
REFERENCE   2  (bases 1 to 7446)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2017 from SnapGene Server 1.1.55
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     repeat_region   1..130
                     /label="AAV2 ITR (alternate)"
                     /note="Functional equivalent of wild-type AAV2 ITR"
     source          1..7213
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(23..34)
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     primer_bind     73..84
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     misc_feature    137..142
                     /Recognition_pattern="T^CTAGA"
                     /annotation_group="TCTAGA"
                     /label="XbaI"
                     /note="Geneious type: restriction site"
     misc_feature    143..148
                     /Recognition_pattern="ATGCA^T"
                     /annotation_group="ATGCAT"
                     /label="NsiI"
                     /note="Geneious type: restriction site"
     primer_bind     143..166
                     /Sequence="ATGCATCATTGGCTCGTGTTCGTG"
                     /Tm="64.5"
                     /%GC="50.0"
                     /Hairpin_Tm="38.3"
                     /Self_Dimer_Tm="None"
                     /created_by="Sabrina"
                     /label="Rosa FLEX mStr Fwd"
     misc_feature    <149..677
                     /note="left homology arm from intron 1 of the mouse
                     Rosa26gene (Soriano, 1999)"
                     /modified_by="Sabrina"
                     /label="HA-L"
     source          <149..>4467
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    700..803
                     /note="adenovirus major late transcript splice acceptor 
                     (Friedrich and Soriano, 1991)"
                     /label="Ad SA"
     intron          1047..1112
                     /note="SV40 (simian virus 40) small t antigen intron"
                     /label="small t intron"
     polyA_signal    1159..1293
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     misc_feature    1409..1414
                     /Recognition_pattern="G^CTAGC"
                     /annotation_group="GCTAGC"
                     /label="NheI"
                     /note="Geneious type: restriction site"
     primer_bind     1409..1438
                     /Sequence="GCTAGCGCTCTAGGAAGAGTACCATTGACG"
                     /Tm="67.9"
                     /%GC="53.3"
                     /Hairpin_Tm="49.3"
                     /Self_Dimer_Tm="16.3"
                     /created_by="Sabrina"
                     /label="CAG mStr Fwd"
     source          complement(<1415..>3417)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1680..1957
                     /label="chicken beta-actin promoter"
     primer_bind     2236..2258
                     /label="pCAGGS-5"
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2320..2339
                     /label="pCAG-F"
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(2374..2407)
                     /label="lox2272"
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (GGATACTT). lox2272 sites are compatible with
                     eachother, but incompatible with loxP or loxN sites."
     protein_bind    complement(2470..2503)
                     /label="loxP"
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (GCATACAT)."
     misc_feature    2518..2523
                     /Recognition_pattern="C^TCGAG"
                     /annotation_group="CTCGAG"
                     /label="XhoI"
                     /note="Geneious type: restriction site"
     gene            complement(2524..>3234)
                     /created_by="User"
                     /modified_by="User"
                     /modified_by="Sabrina"
                     /label="mStrawberry"
     CDS             complement(3213..3234)
                     /created_by="Sabrina"
                     /label="mStrawberry start"
     regulatory      complement(<3235..3240)
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation
                      of translation (Kozak, 1987)"
     misc_feature    3241..3246
                     /Recognition_pattern="GGTAC^C"
                     /annotation_group="GGTACC"
                     /label="KpnI"
                     /note="Geneious type: restriction site"
     protein_bind    3257..3290
                     /label="lox2272"
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (GGATACTT). lox2272 sites are compatible with
                     eachother, but incompatible with loxP or loxN sites."
     protein_bind    3361..3394
                     /label="loxP"
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (GCATACAT)."
     misc_feature    complement(3398..3423)
                     /Sequence="ACGCGTGAATTCGAACCTCTTCGAGG"
                     /Tm="67.1"
                     /%GC="53.8"
                     /Hairpin_Tm="54.0"
                     /Self_Dimer_Tm="12.9"
                     /created_by="Sabrina"
                     /label="CAG mStr Rev"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    3418..3423
                     /Recognition_pattern="A^CGCGT"
                     /annotation_group="ACGCGT"
                     /label="MluI"
                     /note="Geneious type: restriction site"
     primer_bind     complement(3435..3451)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="SK primer"
     primer_bind     complement(3435..3451)
                     /note="For pBluescript vector"
                     /label="pBluescriptSK"
     polyA_signal    3471..3695
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     misc_feature    3724..3729
                     /Recognition_pattern="C^TCGAG"
                     /annotation_group="CTCGAG"
                     /label="XhoI"
                     /note="Geneious type: restriction site"
     promoter        complement(3739..3756)
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     polyA_signal    3761..3895
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     3839..3858
                     /note="SV40 polyA, reverse primer"
                     /label="SV40pA-R"
     misc_feature    3968..>4467
                     /note="right homology arm from intron 1 of the mouse
                     Rosa26 gene (Soriano, 1999)"
                     /label="Rosa26 right arm"
     misc_feature    complement(4448..4467)
                     /Sequence="TGCCAATGCTCTGTCTAGGG"
                     /Hairpin_Tm="38.7"
                     /Tm="59.5"
                     /Self_Dimer_Tm="None"
                     /%GC="55.0"
                     /created_by="primer3"
                     /label="NotI Rosa FLEX mStr rev"
                     /note="Geneious type: primer_bind_reverse"
     misc_feature    4468..4475
                     /Recognition_pattern="GC^GGCCGC"
                     /annotation_group="GCGGCCGC"
                     /label="NotI"
                     /note="Geneious type: restriction site"
     repeat_region   4476..4616
                     /label="AAV2 ITR"
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
     repeat_region   4476..4605
                     /label="AAV2 ITR"
     primer_bind     complement(4522..4533)
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     primer_bind     4583..4594
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     rep_origin      4691..5146
                     /direction="RIGHT"
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4778..4797)
                     /label="F1ori-R"
                     /note="F1 origin, reverse primer"
     primer_bind     4988..5009
                     /label="F1ori-F"
                     /note="F1 origin, forward primer"
     misc_feature    5047..5052
                     /Recognition_pattern="TTA^TAA"
                     /annotation_group="TTATAA"
                     /label="PsiI"
                     /note="Geneious type: restriction site"
     primer_bind     complement(5163..5182)
                     /label="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5282..5304
                     /label="pGEX 3'"
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5342..5360)
                     /label="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5428..5532
                     /gene="bla"
                     /label="bla promoter"
     CDS             5533..6393
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="bla CDS"
     primer_bind     complement(5751..5770)
                     /label="Amp-R"
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6564..7152
                     /direction="RIGHT"
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7053..7072
                     /label="pBR322ori-F"
                     /note="pBR322 origin, forward primer"
ORIGIN      
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gaatgcatca ttggctcgtg ttcgtgcaag ttgagtccat
      181 ccgccggcca gcgggggcgg cgaggaggcg ctcccaggtt ccggccctcc cctcggcccc
      241 gcgccgcaga gtctggccgc gcgcccctgc gcaacgtggc aggaagcgcg cgctgggggc
      301 ggggacgggc agtagggctg agcggctgcg gggcgggtgc aagcacgttt ccgacttgag
      361 ttgcctcaag aggggcgtgc tgagccagac ctccatcgcg cactccgggg agtggaggga
      421 aggagcgagg gctcagttgg gctgttttgg aggcaggaag cacttgctct cccaaagtcg
      481 ctctgagttg ttatcagtaa gggagctgca gtggagtagg cggggagaag gccgcaccct
      541 tctccggagg ggggagggga gtgttgcaat acctttctgg gagttctctg ctgcctcctg
      601 gcttctgagg accgccctgg gcctgggaga atcccttccc cctcttccct cgtgatctgc
      661 aactccagtc tttctaggcg cgcccgggct gcagatctgt agggcgcagt agtccagggt
      721 ttccttgatg atgtcatact tatcctgtcc cttttttttc cacagctcgc ggttgaggac
      781 aaactcttcg cggtctttcc agtggggatc gacggtatcg ataagctggc cgctctagtg
      841 gccgtacggg cccacctgcc gggccactta attaaattta aatcacaatt ccagctgagc
      901 gccggtcgct accattacca gttggtctgg tgtcaaaaat aataataacc gggcaggggg
      961 gatctgcatg gatctttgtg aaggaacctt acttctgtgg tgtgacataa ttggacaaac
     1021 tacctacaga gatttaaagc tctaaggtaa atataaaatt tttaagtgta taatgtgtta
     1081 aactactgat tctaattgtt tgtgtatttt agattccaac ctatggaact gatgaatggg
     1141 agcagtggtg gaatgccaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     1201 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     1261 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     1321 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     1381 ggtatggctg attatgatct gcggccgtgc tagcgctcta ggaagagtac cattgacgtc
     1441 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1501 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1561 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     1621 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1681 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1741 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     1801 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
     1861 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
     1921 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg
     1981 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
     2041 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt
     2101 ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg
     2161 ccctttgtgc ggggggagcg gctcggggct gtccgcgggg ggacggctgc cttcgggggg
     2221 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta
     2281 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc
     2341 tgtctcatca ttttggcaaa gaattggatc cccataactt cgtataaagt atcctatacg
     2401 aagttatatc aaaataggaa gaccaatgct tcaccatcga cccgaattgc caagcatcac
     2461 catcgaccca taacttcgta taatgtatgc tatacgaagt tatactaggg atcccccctc
     2521 gagctacttg tacagctcgt ccatgccgcc ggtggagtgg cggccctcgg cgcgttcgta
     2581 cagttccacg atggtgtagt cctcgttgtg ggaggtgatg tccaacttga tgccgacgat
     2641 gtaggcgccg ggcagctgca cgggcttctt ggccttgtag gtggtcttga cctcagcgtc
     2701 gtagtggccg ccgtccttca gcttcagcct catcttgatc tcgcccttca gggcgccgtc
     2761 ctcggggtac atccgctcgg aggaggcctc ccagcccatg gtcttcttct gcattacggg
     2821 gccgtcggag gggaagttgg tgccgcgcag cttcaccttg tagatgaact cgccgtcctg
     2881 cagggaggag tcctgggtca cggtcaccac gccgccgtcc tcgaagttca tcacgcgctc
     2941 ccacttgaag ccctcgggga aggacagctt caagtagtcg gggatgtcgg cggggtgctt
     3001 cacgtaggcc ttggagccgt aggtgaagtt gggggttagg atgtcccagg cgaagggcag
     3061 ggggccaccc ttggtcacct tcagcttggc ggtctgggtg ccctcgtagg ggcggccctc
     3121 gccctcgccc tcgatctcga actcgtggcc gttcacggag ccctccatgc gcaccttgaa
     3181 gcgcatgaac tccttgatga tggccatgtt attctcctcg cccttgctca ccatggtggc
     3241 ggtaccgaat tctagtataa cttcgtatag gatactttat acgaagttat cattgggatt
     3301 cttcctattt tgatccaagc atcaccatcg accctctagt ccagatctca ccatcgaccc
     3361 ataacttcgt atagcataca ttatacgaag ttatgtccct cgaagaggtt cgaattcacg
     3421 cgtaaatgat tgcagatcca ctagttctag agctcgctga tcagcctcga ctgtgccttc
     3481 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3541 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3601 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3661 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3721 gggctcgagc ctctagaact atagtgagtc gtattacgta gatccagaca tgataagata
     3781 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     3841 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     3901 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt
     3961 cgcggcccta gaagatgggc gggagtcttc tgggcaggct taaaggctaa cctggtgtgt
     4021 gggcgttgtc ctgcagggga attgaacagg tgtaaaattg gagggacaag acttcccaca
     4081 gattttcggt tttgtcggga agttttttaa taggggcaaa taaggaaaat gggaggatag
     4141 gtagtcatct ggggttttat gcagcaaaac tacaggttat tattgcttgt gatccgcctc
     4201 ggagtatttt ccatcgaggt agattaaaga catgctcacc cgagttttat actctcctgc
     4261 ttgagatcct tactacagta tgaaattaca gtgtcgcgag ttagactatg taagcagaat
     4321 tttaatcatt tttaaagagc ccagtacttc atatccattt ctcccgctcc ttctgcagcc
     4381 ttatcaaaag gtattttaga acactcattt tagccccatt ttcatttatt atactggctt
     4441 atccaacccc tagacagagc attggcagcg gccgcaggaa cccctagtga tggagttggc
     4501 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4561 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4621 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4681 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4741 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     4801 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4861 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4921 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4981 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     5041 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     5101 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5161 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5221 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5281 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5341 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5401 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5461 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5521 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5581 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5641 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5701 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5761 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5821 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5881 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5941 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6001 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6061 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6121 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6181 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6241 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6301 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6361 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6421 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6481 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6541 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6601 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6661 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6721 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6781 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6841 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6901 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6961 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7021 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7081 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7141 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7201 tttgctcaca tgt
//
