LOCUS       sequence-317325-        7727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Rosa26.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7727)
  TITLE     Zweifel CRISPR AAV Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7727)
  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            "Unpublished"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: pAAV-CMV-FLEXfrt-SaCas9-U6-sgRosa26
FEATURES             Location/Qualifiers
     source          1..7727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    627..660
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(732..812)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(819..866)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATRKAGQAKKKK"
     CDS             complement(867..4022)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             complement(4047..4067)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4257..4274)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4263..4470
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4477..4725
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4477..4497
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4648..4667
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4748..4823
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4837..4977
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4837..4966
                     /label=AAV2 ITR
     rep_origin      5052..5507
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5139..5158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5349..5370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5524..5543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5643..5665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5703..5721)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5789..5893
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5894..5962,5963..6754)
                     /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     complement(6112..6131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6925..7513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7414..7433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7575..7704
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactaccg
      601 gtgctagcgt cgacagaatt ggatccgaag ttcctattct ctagaaagta taggaacttc
      661 caaggcctga agttcctatt cttcaaaagg tataggaact tcgcggccgc ttgatatctt
      721 ggcgcgcctt aagcgtaatc tggaacatcg tatgggtaag cgtaatctgg aacatcgtat
      781 gggtaagcgt aatctggaac atcgtatggg taggatccct ttttcttttt tgcctggccg
      841 gcctttctcg tggccgccgg ccttttgccc tttttgatga tctgagggtg cttcttagat
      901 ttcacttcat acaggttgcc cagaatgtct gtgctgtact tcttaatgct ctgggtcttg
      961 gaggcgattg tcttaatgat cctggggggc ctcttgtcgt tcatgttttc caggtactcg
     1021 cggtaggtga tgtcgatcat gttcacttcg atccggttca gcaggtcgtt gttcacgccg
     1081 atcactctat acagctcgcc gttgatcttg atcagatcgt tgttgtagaa ggaggcgata
     1141 aactcggcct ggttgctgat cttcttcagc ttcttagctt cctcatagca cttgctattc
     1201 acttcgtagt agttttcttt tttgatcaca tccagattct tcacggtcac gaacttgtac
     1261 acgccattgt ccaggtacac gtcgaatctg tagggcttca gggacagctt cacgaccttg
     1321 tttctgctgt tggggtagtc gtcggtgatg tccagatggg cgttcagttt gttgccgtaa
     1381 tacttaatct tcttgatcac ggggccgttg tcctttttgg agtacttggt caggtagttc
     1441 ccggtttcct cgtagtactt gtacagggga ttcttctcgt cgccgtactg ttccataatc
     1501 agcttcagtt tctggtaggt ctgggggtcg tggtggtaca tcagcagctt ttcggggctc
     1561 ttgttgatca gctttttcag cttgtcattg tccttgtcgt acaggccgtt cagattgttc
     1621 acgatcaggg tgttgccctt gtcgtccttc cgggtggagt acagggtgtc gttaatcagc
     1681 tctctattag gcttcttgtc cacccggtgg ctgtacttgt agtccttgaa gtccttaatg
     1741 tgcttgatct ggtggggggt gatgaagatc tctttgtact cctgctcggt ttcgatctcg
     1801 ggcatgctct cggcctgctt ttcctcgaac atctggtttt ccatcacttt tttggccttg
     1861 tccagtttct tccactcttt gaagatgaaa tcggcgttgg caatgatcag ggcgtcctcg
     1921 gcgtggtgct tgtacccctt gttccgctct ttcttaaact tccacttccg ccgcagaaag
     1981 ctggtgaagc cgccattgat ggacttcact ttcacgtcca ggttgttcac tctgaagtag
     2041 ctccgcagca ggttcatcag gcctctggtg gcgtatctgg tatccaccag gttccggttg
     2101 atgaagtctt tctgcacgga gaacctgttg atgtcccgtt cttccagcag atactctttc
     2161 ttggtcttgc tgattctgcc cttgcccttg gccagattca ggatgtgctt cttgaaggtt
     2221 tcgtagctga tcttgctgtc gctgctgctc aggtactgga atggggtccg gttgcccttc
     2281 ttgctgtttt cttcctgctt cacgagcacc ttgttgttga agctgttgtc gaaggacacg
     2341 cttctgggga tgatgtggtc cacctcatag ttgaaggggt tgttcagcag atcttccaga
     2401 gggatggctt ccaggctgta caggcacttg ccttcctgca tgtcgtgcag cttgatcttc
     2461 tcgatcaggt acttggcgtt ctctttgccg gtggtccgga tgatttcctc gatccgctcg
     2521 ttggtctgcc ggttccgctt ctgcatctcg ttgatcattt tctgggcgtc cttggagttc
     2581 ttctcgcggg ccagctcgat aatgatgtcg ttgggcaggc cgtacttctt gatgatggcg
     2641 ttgatcactt tgatgctctg gatgaagctt ctcttcacga cggggctcag gatgaagtcg
     2701 tccaccaggg tggtggggat ctctttctgc tgggacaggt ccaccttctt gggcaccagc
     2761 ttcagccggt tgaagatagc gatctggttg tcgttggtgt gccacagctc gtccaggatc
     2821 aggttgatgg ccttcaggct caggttgtgg gtgccggtat agcccttcag attagagatc
     2881 tgctcgatct cttcctgggt cagctcggag ttcagattgg tcagttcttc ctggatgtcc
     2941 tcgctgctct ggtagatggt caggatcttg gcaatctgat ccagcagctc ggcgttctca
     3001 ataatctctt tccgggcggt aatgtccttg atgtcgtggt acaccttcag gttggtgaac
     3061 tcgggcttgc cggtgctggt cactctgtag cccttaatat cctcttcgtt cacgaggatt
     3121 tctttggcga tctgcttcag ggtgggcttc ttcttctgct tgaacacgtt ctcgatgatc
     3181 tggaacttct cgtaatattc cagcttctcg ttctcgtccc tggtgatcac gagattgttc
     3241 aggtcgttca gggcgttgta caggtcggcg ttgtaggcgt acttcacgct ccgcagttcc
     3301 tcggggaagt aggtgcagtg gcccatcagc atctcgtacc attctttgat gtccttccag
     3361 ccgaaggggc tgccctcgcc aggtccctca tagtaggtcc gccgggtttc cagcaggtcg
     3421 atgtaggtgt cgatgaagct ctggtccagc tggtggtagg ccttctgcac cttcagcagc
     3481 tgtttggctt ctttcacgta gtcgctggtc ttgaatctgt tgatgctgcc ccgcacttcg
     3541 ccgtctttct tcagccgttc cagctgcagt tcggccacgt atttctcttc cagggccttg
     3601 ctgttccggc tgatctgctc tttggtggac agctcgttgc cggtgtcctc ttccacctcg
     3661 ttcacgttgt gcacgcctct tctcttggcc aggtgcagca gggcggcaga gaactcttcc
     3721 tcgctcagct tctggctcag gcccttcact ctggcctcgt aggggttgat gccgctcagc
     3781 tcgctgtggt cggtcagcag gttgtagtcg aacagcagct tcttcactct ctggattcta
     3841 tgccgcctcc gccgcttcag ccttctggcg cctctcttgc tccgcctgcc ctcgttgttt
     3901 tccacgttgg cctctttgaa cagccgcacg ccggcatcga tcacgtcccg tgtctcgtag
     3961 tcgatgatgc cgtagcccac gctggtgatg ccgatgtcca ggcccaggat gtagttccgc
     4021 ttggctgctg ggactccgtg gataccgacc ttccgcttct tctttggggc catgcggccg
     4081 ctgcagaccg gtttagatct ttactagttt aagcttcaat tggaagttcc tattctttct
     4141 agagtatagg aacttcctgc agtaggctag cgaagttcct attccttttg aagtatagga
     4201 acttcgtcga gggggggccc gagcttatcg atgaattcct agagctcgct gatcagcctc
     4261 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4321 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4381 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4441 ttgggaagag aatagcaggc atgctgggga ggtaccgagg gcctatttcc catgattcct
     4501 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     4561 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     4621 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     4681 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgctcg atggaaaata
     4741 ctccgaggtt ttagtactct ggaaacagaa tctactaaaa caaggcaaaa tgccgtgttt
     4801 atctcgtcaa cttgttggcg agatttttgc ggccgcagga acccctagtg atggagttgg
     4861 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     4921 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     4981 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     5041 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5101 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     5161 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5221 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     5281 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5341 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     5401 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     5461 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     5521 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     5581 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5641 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     5701 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     5761 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5821 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5881 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5941 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     6001 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6061 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     6121 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6181 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6241 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6301 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6361 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6421 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6481 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6541 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6601 tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6661 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6721 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6781 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6841 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6901 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6961 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7021 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     7081 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7141 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7201 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7261 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7321 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7381 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7441 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7501 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7561 ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc
     7621 gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg
     7681 ccaactccat cactaggggt tcctgcggcc tctagactcg aggcgtt
//
