LOCUS       sequence-269796-        7219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA against the right loxP site of the Ai9 allele.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7219)
  TITLE     Ai9-Sa-gRNAs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7219)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: 1782_pAAV-U6-Ai9-Sa-gRNA2-CB-SACas9-HA-OLLAS-s...
FEATURES             Location/Qualifiers
     source          1..7219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   20..149
                     /label=AAV2 ITR
     promoter        271..519
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     271..291
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     442..461
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        541..616
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     CDS             1103..1123
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4304..4351
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4358..4384
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4385..4426
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     polyA_signal    4444..4492
                     /note="synthetic polyadenylation signal"
     repeat_region   4540..4669
                     /label=AAV2 ITR
     rep_origin      complement(4962..5550)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5042..5061)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5721..6512,6513..6581))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6344..6363
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6582..6686)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6712..7167)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6849..6870)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7061..7080
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctcttcgcta ttacgccagc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
       61 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      121 agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc catgctactt
      181 atctacgtag ccatgctctg gtgtacaaaa aagcaggctt taaaggaacc aattcagtcg
      241 actggatccg gtaccaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat
      301 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
      361 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
      421 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
      481 ttcttggctt tatatatctt gtggaaagga cgaaacaccg acgaagttat attaagggtt
      541 gttttagtac tctggaaaca gaatctacta aaacaaggca aaatgccgtg tttatctcgt
      601 caacttgttg gcgagatttt tttctagacc cagctttcaa gcttgattaa cccgccatgc
      661 tacttatcta cgtagccatg ctctaggaag atcggaattc gcccttaagc tagtatgcca
      721 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      781 atgaccttat gggactttcc tacttggcag tacatctact cgaggccacg ttctgcttca
      841 ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat
      901 tttgtgcagc gatgggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc
      961 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
     1021 ctataaaaag cgaagcgcgc ggcgggcggg agcgggatca gaatgatctg atatcatcga
     1081 tgaattcgag ctcaccatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc
     1141 agcagccaag cggaactaca tcctgggcct ggacatcggc atcaccagcg tgggctacgg
     1201 catcatcgac tacgagacac gggacgtgat cgatgccggc gtgcggctgt tcaaagaggc
     1261 caacgtggaa aacaacgagg gcaggcggag caagagaggc gccagaaggc tgaagcggcg
     1321 gaggcggcat agaatccaga gagtgaagaa gctgctgttc gactacaacc tgctgaccga
     1381 ccacagcgag ctgagcggca tcaaccccta cgaggccaga gtgaagggcc tgagccagaa
     1441 gctgagcgag gaagagttct ctgccgccct gctgcacctg gccaagagaa gaggcgtgca
     1501 caacgtgaac gaggtggaag aggacaccgg caacgagctg tccaccagag agcagatcag
     1561 ccggaacagc aaggccctgg aagagaaata cgtggccgaa ctgcagctgg aacggctgaa
     1621 gaaagacggc gaagtgcggg gcagcatcaa cagattcaag accagcgact acgtgaaaga
     1681 agccaaacag ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga gcttcatcga
     1741 cacctacatc gacctgctgg aaacccggcg gacctactat gagggacctg gcgagggcag
     1801 ccccttcggc tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc actgcaccta
     1861 cttccccgag gaactgcgga gcgtgaagta cgcctacaac gccgacctgt acaacgccct
     1921 gaacgacctg aacaatctcg tgatcaccag ggacgagaac gagaagctgg aatattacga
     1981 gaagttccag atcatcgaga acgtgttcaa gcagaagaag aagcccaccc tgaagcagat
     2041 cgccaaagaa atcctcgtga acgaagagga tattaagggc tacagagtga ccagcaccgg
     2101 caagcccgag ttcaccaacc tgaaggtgta ccacgacatc aaggacatta ccgcccggaa
     2161 agagattatt gagaacgccg agctgctgga tcagattgcc aagatcctga ccatctacca
     2221 gagcagcgag gacatccagg aagaactgac caatctgaac tccgagctga cccaggaaga
     2281 gatcgagcag atctctaatc tgaagggcta taccggcacc cacaacctga gcctgaaggc
     2341 catcaacctg atcctggacg agctgtggca caccaacgac aaccagatcg ctatcttcaa
     2401 ccggctgaag ctggtgccca agaaggtgga cctgtcccag cagaaagaga tccccaccac
     2461 cctggtggac gacttcatcc tgagccccgt cgtgaagaga agcttcatcc agagcatcaa
     2521 agtgatcaac gccatcatca agaagtacgg cctgcccaac gacatcatta tcgagctggc
     2581 ccgcgagaag aactccaagg acgcccagaa aatgatcaac gagatgcaga agcggaaccg
     2641 gcagaccaac gagcggatcg aggaaatcat ccggaccacc ggcaaagaga acgccaagta
     2701 cctgatcgag aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt acagcctgga
     2761 agccatccct ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg accacatcat
     2821 ccccagaagc gtgtccttcg acaacagctt caacaacaag gtgctcgtga agcaggaaga
     2881 aaacagcaag aagggcaacc ggaccccatt ccagtacctg agcagcagcg acagcaagat
     2941 cagctacgaa accttcaaga agcacatcct gaatctggcc aagggcaagg gcagaatcag
     3001 caagaccaag aaagagtatc tgctggaaga acgggacatc aacaggttct ccgtgcagaa
     3061 agacttcatc aaccggaacc tggtggatac cagatacgcc accagaggcc tgatgaacct
     3121 gctgcggagc tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca tcaatggcgg
     3181 cttcaccagc tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca aggggtacaa
     3241 gcaccacgcc gaggacgccc tgatcattgc caacgccgat ttcatcttca aagagtggaa
     3301 gaaactggac aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa ggcaggccga
     3361 gagcatgccc gagatcgaaa ccgagcagga gtacaaagag atcttcatca ccccccacca
     3421 gatcaagcac attaaggact tcaaggacta caagtacagc caccgggtgg acaagaagcc
     3481 taatagagag ctgattaacg acaccctgta ctccacccgg aaggacgaca agggcaacac
     3541 cctgatcgtg aacaatctga acggcctgta cgacaaggac aatgacaagc tgaaaaagct
     3601 gatcaacaag agccccgaaa agctgctgat gtaccaccac gacccccaga cctaccagaa
     3661 actgaagctg attatggaac agtacggcga cgagaagaat cccctgtaca agtactacga
     3721 ggaaaccggg aactacctga ccaagtactc caaaaaggac aacggccccg tgatcaagaa
     3781 gattaagtat tacggcaaca aactgaacgc ccatctggac atcaccgacg actaccccaa
     3841 cagcagaaac aaggtcgtga agctgtccct gaagccctac agattcgacg tgtacctgga
     3901 caatggcgtg tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa aagaaaacta
     3961 ctacgaagtg aatagcaagt gctatgagga agctaagaag ctgaagaaga tcagcaacca
     4021 ggccgagttt atcgcctcct tctacaacaa cgatctgatc aagatcaacg gcgagctgta
     4081 tagagtgatc ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca tgatcgacat
     4141 cacctaccgc gagtacctgg aaaacatgaa cgacaagagg ccccccagga tcattaagac
     4201 aatcgcctcc aagacccaga gcattaagaa gtacagcaca gacattctgg gcaacctgta
     4261 tgaagtgaaa tctaagaagc accctcagat catcaaaaag ggcaaaaggc cggcggccac
     4321 gaaaaaggcc ggccaggcaa aaaagaaaaa gggatcctac ccatacgatg ttccagatta
     4381 cgctagcggc ttcgccaacg agcttggacc caggttgatg ggaaagtaag aattcctaga
     4441 gcgaataaaa gatctttatt ttcattagat ctgtgtgttg gttttttgtg tgatgcagct
     4501 acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt
     4561 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4621 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg
     4681 aatcggccaa cgcgcgggga gaggcgggag cgagcgagcg cgcagctgca ttaatgaatc
     4741 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4801 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4861 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4921 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4981 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5041 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5101 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5161 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5221 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5281 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5341 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5401 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5461 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5521 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5581 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5641 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5701 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5761 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5821 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5881 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5941 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6001 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6061 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6121 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6181 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6241 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6301 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6361 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6421 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6481 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6541 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6601 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6661 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     6721 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     6781 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     6841 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     6901 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     6961 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     7021 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     7081 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     7141 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     7201 aagggcgatc ggtgcgggc
//
