LOCUS       sequence-269807-        7465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA against the left loxP site of the Ai9 allele.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7465)
  TITLE     Ai9-Sa-gRNAs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7465)
  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            "Unpublished"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: 1781_pAAV-U6-Ai9-Sa-gRNA1-CB-SACas9-HA-OLLAS-s...
FEATURES             Location/Qualifiers
     source          1..7465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        252..500
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     252..272
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     423..442
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        522..597
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     CDS             1084..1104
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4285..4332
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4339..4365
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4366..4407
                     /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    4425..4473
                     /note="synthetic polyadenylation signal"
     repeat_region   4521..4650
                     /label=AAV2 ITR
     primer_bind     complement(4787..4803)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4787..4803)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4800..4822)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4811..4827
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4835..4841,4842..4859,4860..4865))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4880..4901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5189..5777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5269..5288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5948..6739,6740..6808))
                     /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     6571..6590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6809..6913)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6939..7394)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7076..7097)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7288..7307
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 ggtgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg
      241 tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      301 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
      361 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      421 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      481 tgtggaaagg acgaaacacc gctctagagt cgcagatcct cgttttagta ctctggaaac
      541 agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt
      601 ttttctagac ccagctttca agcttgatta acccgccatg ctacttatct acgtagccat
      661 gctctaggaa gatcggaatt cgcccttaag ctagtatgcc aagtacgccc cctattgacg
      721 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      781 ctacttggca gtacatctac tcgaggccac gttctgcttc actctcccca tctccccccc
      841 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      901 gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
      961 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
     1021 cggcgggcgg gagcgggatc agaatgatct gatatcatcg atgaattcga gctcaccatg
     1081 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccaa gcggaactac
     1141 atcctgggcc tggacatcgg catcaccagc gtgggctacg gcatcatcga ctacgagaca
     1201 cgggacgtga tcgatgccgg cgtgcggctg ttcaaagagg ccaacgtgga aaacaacgag
     1261 ggcaggcgga gcaagagagg cgccagaagg ctgaagcggc ggaggcggca tagaatccag
     1321 agagtgaaga agctgctgtt cgactacaac ctgctgaccg accacagcga gctgagcggc
     1381 atcaacccct acgaggccag agtgaagggc ctgagccaga agctgagcga ggaagagttc
     1441 tctgccgccc tgctgcacct ggccaagaga agaggcgtgc acaacgtgaa cgaggtggaa
     1501 gaggacaccg gcaacgagct gtccaccaga gagcagatca gccggaacag caaggccctg
     1561 gaagagaaat acgtggccga actgcagctg gaacggctga agaaagacgg cgaagtgcgg
     1621 ggcagcatca acagattcaa gaccagcgac tacgtgaaag aagccaaaca gctgctgaag
     1681 gtgcagaagg cctaccacca gctggaccag agcttcatcg acacctacat cgacctgctg
     1741 gaaacccggc ggacctacta tgagggacct ggcgagggca gccccttcgg ctggaaggac
     1801 atcaaagaat ggtacgagat gctgatgggc cactgcacct acttccccga ggaactgcgg
     1861 agcgtgaagt acgcctacaa cgccgacctg tacaacgccc tgaacgacct gaacaatctc
     1921 gtgatcacca gggacgagaa cgagaagctg gaatattacg agaagttcca gatcatcgag
     1981 aacgtgttca agcagaagaa gaagcccacc ctgaagcaga tcgccaaaga aatcctcgtg
     2041 aacgaagagg atattaaggg ctacagagtg accagcaccg gcaagcccga gttcaccaac
     2101 ctgaaggtgt accacgacat caaggacatt accgcccgga aagagattat tgagaacgcc
     2161 gagctgctgg atcagattgc caagatcctg accatctacc agagcagcga ggacatccag
     2221 gaagaactga ccaatctgaa ctccgagctg acccaggaag agatcgagca gatctctaat
     2281 ctgaagggct ataccggcac ccacaacctg agcctgaagg ccatcaacct gatcctggac
     2341 gagctgtggc acaccaacga caaccagatc gctatcttca accggctgaa gctggtgccc
     2401 aagaaggtgg acctgtccca gcagaaagag atccccacca ccctggtgga cgacttcatc
     2461 ctgagccccg tcgtgaagag aagcttcatc cagagcatca aagtgatcaa cgccatcatc
     2521 aagaagtacg gcctgcccaa cgacatcatt atcgagctgg cccgcgagaa gaactccaag
     2581 gacgcccaga aaatgatcaa cgagatgcag aagcggaacc ggcagaccaa cgagcggatc
     2641 gaggaaatca tccggaccac cggcaaagag aacgccaagt acctgatcga gaagatcaag
     2701 ctgcacgaca tgcaggaagg caagtgcctg tacagcctgg aagccatccc tctggaagat
     2761 ctgctgaaca accccttcaa ctatgaggtg gaccacatca tccccagaag cgtgtccttc
     2821 gacaacagct tcaacaacaa ggtgctcgtg aagcaggaag aaaacagcaa gaagggcaac
     2881 cggaccccat tccagtacct gagcagcagc gacagcaaga tcagctacga aaccttcaag
     2941 aagcacatcc tgaatctggc caagggcaag ggcagaatca gcaagaccaa gaaagagtat
     3001 ctgctggaag aacgggacat caacaggttc tccgtgcaga aagacttcat caaccggaac
     3061 ctggtggata ccagatacgc caccagaggc ctgatgaacc tgctgcggag ctacttcaga
     3121 gtgaacaacc tggacgtgaa agtgaagtcc atcaatggcg gcttcaccag ctttctgcgg
     3181 cggaagtgga agtttaagaa agagcggaac aaggggtaca agcaccacgc cgaggacgcc
     3241 ctgatcattg ccaacgccga tttcatcttc aaagagtgga agaaactgga caaggccaaa
     3301 aaagtgatgg aaaaccagat gttcgaggaa aggcaggccg agagcatgcc cgagatcgaa
     3361 accgagcagg agtacaaaga gatcttcatc accccccacc agatcaagca cattaaggac
     3421 ttcaaggact acaagtacag ccaccgggtg gacaagaagc ctaatagaga gctgattaac
     3481 gacaccctgt actccacccg gaaggacgac aagggcaaca ccctgatcgt gaacaatctg
     3541 aacggcctgt acgacaagga caatgacaag ctgaaaaagc tgatcaacaa gagccccgaa
     3601 aagctgctga tgtaccacca cgacccccag acctaccaga aactgaagct gattatggaa
     3661 cagtacggcg acgagaagaa tcccctgtac aagtactacg aggaaaccgg gaactacctg
     3721 accaagtact ccaaaaagga caacggcccc gtgatcaaga agattaagta ttacggcaac
     3781 aaactgaacg cccatctgga catcaccgac gactacccca acagcagaaa caaggtcgtg
     3841 aagctgtccc tgaagcccta cagattcgac gtgtacctgg acaatggcgt gtacaagttc
     3901 gtgaccgtga agaatctgga tgtgatcaaa aaagaaaact actacgaagt gaatagcaag
     3961 tgctatgagg aagctaagaa gctgaagaag atcagcaacc aggccgagtt tatcgcctcc
     4021 ttctacaaca acgatctgat caagatcaac ggcgagctgt atagagtgat cggcgtgaac
     4081 aacgacctgc tgaaccggat cgaagtgaac atgatcgaca tcacctaccg cgagtacctg
     4141 gaaaacatga acgacaagag gccccccagg atcattaaga caatcgcctc caagacccag
     4201 agcattaaga agtacagcac agacattctg ggcaacctgt atgaagtgaa atctaagaag
     4261 caccctcaga tcatcaaaaa gggcaaaagg ccggcggcca cgaaaaaggc cggccaggca
     4321 aaaaagaaaa agggatccta cccatacgat gttccagatt acgctagcgg cttcgccaac
     4381 gagcttggac ccaggttgat gggaaagtaa gaattcctag agcgaataaa agatctttat
     4441 tttcattaga tctgtgtgtt ggttttttgt gtgatgcagc tacgtagata agtagcatgg
     4501 cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg
     4561 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4621 ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat gaatcggcca acgcgcgggg
     4681 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4741 gtcgttcggc tgcggcgagc ggtatcagct gcttggcgta atcatggtca tagctgtttc
     4801 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4861 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4921 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4981 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5041 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5101 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5161 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5221 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5281 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5341 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5401 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5461 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5521 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5581 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5641 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5701 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5761 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5821 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5881 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5941 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6001 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6061 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6121 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6181 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6241 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6301 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6361 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6421 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6481 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6541 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6601 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6661 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6721 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6781 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6841 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6901 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     6961 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     7021 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     7081 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     7141 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     7201 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     7261 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     7321 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     7381 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     7441 gcgggcctct tcgctattac gccag
//
