LOCUS       sequence-343382-        5762 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Plasmid containing separate SpCas9 gRNAs for targeted excision of 
            inserted STOP Cassette upstream of reporter alleles found in Ai14 
            and Ai6 mice. ITRs flank the cassette for easy vector creation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5762)
  AUTHORS   Lang JF, Toulmin SA, Brida KL, Eisenlohr LC, Davidson BL
  TITLE     Standard screening methods underreport AAV-mediated transduction and
            gene editing.
  JOURNAL   Nat Commun. 2019 Jul 30;10(1):3415. doi: 10.1038/s41467-019-11321-7.
  PUBMED    31363095
REFERENCE   2  (bases 1 to 5762)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-07-30"; volume: "10(1):3415. doi"; pages: " 
            10.1038/s41467-019-11321-7"
COMMENT     Sequence Label: pFB_ROSA26_SpCas9_gRNAs
FEATURES             Location/Qualifiers
     source          1..5762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(493..1284,1285..1353))
                     /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     1116..1135
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1354..1458)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1484..1938)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1621..1642)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1832..1851
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     mobile_element  2122..2287
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     polyA_signal    2316..2450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2340..2359)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2394..2413
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2617..2757
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2628..2757
                     /label=AAV2 ITR
     promoter        2909..3157
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3178..3253
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3270..3518
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3540..3615
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   3725..3854
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(4194..4199,4200..4216,4217..4222)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             4367..4900
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     mobile_element  4967..5191
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     rep_origin      complement(join(5496..5762,1..322))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5576..5595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
       61 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
      121 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
      181 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
      241 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
      301 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
      361 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
      421 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
      481 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
      541 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
      601 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
      661 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
      721 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
      781 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
      841 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
      901 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
      961 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     1021 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     1081 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     1141 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     1201 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     1261 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     1321 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     1381 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     1441 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     1501 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     1561 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     1621 ccagtttgga acaagagtcc actattaaga acgtggactc caacgtcaaa gggcgaaaaa
     1681 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     1741 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     1801 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     1861 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     1921 cgccgctaca gggcgcgtcc cattcgccat tcaggctgca aataagcgtt gatattcagt
     1981 caattacaaa cattaataac gaagagatga cagaaaaatt ttcattctgt gacagagaaa
     2041 aagtagccga agatgacggt ttgtcacatg gagttggcag gatgtttgat taaaaacata
     2101 acaggaagaa aaatgccccg ctgtgggcgg acaaaatagt tgggaactgg gaggggtgga
     2161 aatggagttt ttaaggatta tttagggaag agtgacaaaa tagatgggaa ctgggtgtag
     2221 cgtcgtaagc taatacgaaa attaaaaatg acaaaatagt ttggaactag atttcactta
     2281 tctggttcgg atctcctagg cgatatcagt gatcagatcc agacatgata agatacattg
     2341 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     2401 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     2461 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     2521 aaaacctcta caaatgtggt atggctgatt atgatcctct agtacttctc gacaagctta
     2581 cattattgaa gcatttatca gggttattgt ctcagacctg caggcagctg cgcgctcgct
     2641 cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct
     2701 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttccttgt
     2761 agttaatgat taacccgcca tgctacttat ctacgtagcc atgctctaga gcggccgcac
     2821 gcgtactagt aagggcctcg aacgcgtgtc tgtctgcaca tttcgtagag cgagtgttcc
     2881 gatactctaa tctccctagg actagagaga gggcctattt cccatgattc cttcatattt
     2941 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
     3001 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     3061 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     3121 cttggcttta tatatcttgt ggaaaggacg aaacaccgta tgctatacga agttattgtt
     3181 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     3241 accgagtcgg tgctttttta ggtttaaacg agggcctatt tcccatgatt ccttcatatt
     3301 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
     3361 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     3421 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     3481 tcttggcttt atatatcttg tggaaaggac gaaacaccga aagaattgat ttgataccgg
     3541 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     3601 gcaccgagtc ggtgcttttt ttctgtgtgt tggttttttg tgtgtgcgga ccgagcggac
     3661 cgagcggccg ctctagagca tggctacgta gataagtagc atggcgggtt aatcattaac
     3721 tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     3781 gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg
     3841 cagctgcctg caggcatgca agctgtagcc aaccactaga actatagcta gagtcctggg
     3901 cgaacaaacg atgctcgcct tccagaaaac cgaggatgcg aaccacttca tccggggtca
     3961 gcaccaccgg caagcgccgc gacggccgag gtcttccgat ctcctgaagc cagggcagat
     4021 ccgtgcacag caccttgccg tagaagaaca gcaaggccgc caatgcctga cgatgcgtgg
     4081 agaccgaaac cttgcgctcg ttcgccagcc aggacagaaa tgcctcgact tcgctgctgc
     4141 ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc cagttgacat
     4201 aagcctgttc ggttcgtaaa ctgtaatgca agtagcgtat gcgctcacgc aactggtcca
     4261 gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg
     4321 actgtttttt tgtacagtct atgcctcggg catccaagca gcaacgatgt tacgcagcag
     4381 caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaggtggct caagtatggg
     4441 catcattcgc acatgtaggc tcggccctga ccaagtcaaa tccatgcggg ctgctcttga
     4501 tcttttcggt cgtgagttcg gagacgtagc cacctactcc caacatcagc cggactccga
     4561 ttacctcggg aacttgctcc gtagtaagac attcatcgcg cttgctgcct tcgaccaaga
     4621 agcggttgtt ggcgctctcg cggcttacgt tctgcccaag tttgagcagc cgcgtagtga
     4681 gatctatatc tatgatctcg cagtctccgg cgagcaccgg aggcagggca ttgccaccgc
     4741 gctcatcaat ctcctcaagc atgaggccaa cgcgcttggt gcttatgtga tctacgtgca
     4801 agcagattac ggtgacgatc ccgcagtggc tctctataca aagttgggca tacgggaaga
     4861 agtgatgcac tttgatatcg acccaagtac cgccacctaa caattcgttc aagccgagat
     4921 cggcttcccg gccgcggagt tgttcggtaa attgtcacaa cgccgcgaat atagtcttta
     4981 ccatgccctt ggccacgccc ctctttaata cgacgggcaa tttgcacttc agaaaatgaa
     5041 gagtttgctt tagccataac aaaagtccag tatgcttttt cacagcataa ctggactgat
     5101 ttcagtttac aactattctg tctagtttaa gactttattg tcatagttta gatctatttt
     5161 gttcagttta agactttatt gtccgcccac acccgcttac gcagggcatc catttattac
     5221 tcaaccgtaa ccgattttgc caggttacgc ggctggtcta tgcggtgtga aataccgcac
     5281 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     5341 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5401 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5461 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5521 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5581 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5641 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5701 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5761 cc
//
