LOCUS       sequence-386494-        5662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV plasmid expressing the FlpO recombinase. with empty gRNA .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5662)
  TITLE     Cas9/CRISPR pig transposon 
            plasmid_LIR_TRE_CAG_mKateII-Puro-STOP_mVenus-Cas9-PA-RIR
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5662)
  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            "Unpublished"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: AAV:ITR-U6-sgRNA (Backbone) PCB-FlPO-WPRE-synt...
FEATURES             Location/Qualifiers
     source          1..5662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        577..862
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1272..1499
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1511..1520
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1517..1522,1523..1543,1544..2812)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     CDS             2813..2839
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     repeat_region   2925..3065
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2925..3054
                     /label=AAV2 ITR
     rep_origin      3140..3595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3227..3246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3437..3458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3612..3631)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3731..3753
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3791..3809)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3877..3981
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3982..4050,4051..4842)
                     /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(4200..4219)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5013..5601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5502..5521
                     /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 gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgtagcg
      541 cgtgcgccaa ttctgcagac aaattctaga ggtacccgtt acataactta cggtaaatgg
      601 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
      661 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      721 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      781 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      841 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      901 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      961 gatgggggcg gggggggggg ggcgcgcgcc aggcggggcg gggggggggg gggggggggg
     1021 gggggggggg gggggggggg gggggggggg sgragggggg gggggggsgg gggggkkggk
     1081 kgggkggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggggsggg
     1141 gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc
     1201 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
     1261 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
     1321 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
     1381 gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag ggatggttgg
     1441 ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact ttttttcagg
     1501 ttggaccggt gccaccatgg ctcctaagaa gaagaggaag gtgatgagcc agttcgacat
     1561 cctgtgcaag acccccccca aggtgctggt gcggcagttc gtggagagat tcgagaggcc
     1621 cagcggcgag aagatcgcca gctgtgccgc cgagctgacc tacctgtgct ggatgatcac
     1681 ccacaacggc accgccatca agagggccac cttcatgagc tacaacacca tcatcagcaa
     1741 cagcctgagc ttcgacatcg tgaacaagag cctgcagttc aagtacaaga cccagaaggc
     1801 caccatcctg gaggccagcc tgaagaagct gatccccgcc tgggagttca ccatcatccc
     1861 ttacaacggc cagaagcacc agagcgacat caccgacatc gtgtccagcc tgcagctgca
     1921 gttcgagagc agcgaggagg ccgacaaggg caacagccac agcaagaaga tgctgaaggc
     1981 cctgctgtcc gagggcgaga gcatctggga gatcaccgag aagatcctga acagcttcga
     2041 gtacaccagc aggttcacca agaccaagac cctgtaccag ttcctgttcc tggccacatt
     2101 catcaactgc ggcaggttca gcgacatcaa gaacgtggac cccaagagct tcaagctggt
     2161 gcagaacaag tacctgggcg tgatcattca gtgcctggtg accgagacca agacaagcgt
     2221 gtccaggcac atctactttt tcagcgccag aggcaggatc gaccccctgg tgtacctgga
     2281 cgagttcctg aggaacagcg agcccgtgct gaagagagtg aacaggaccg gcaacagcag
     2341 cagcaacaag caggagtacc agctgctgaa ggacaacctg gtgcgcagct acaacaaggc
     2401 cctgaagaag aacgccccct accccatctt cgctatcaag aacggcccta agagccacat
     2461 cggcaggcac ctgatgacca gctttctgag catgaagggc ctgaccgagc tgacaaacgt
     2521 ggtgggcaac tggagcgaca agagggcctc cgccgtggcc aggaccacct acacccacca
     2581 gatcaccgcc atccccgacc actacttcgc cctggtgtcc aggtactacg cctacgaccc
     2641 catcagcaag gagatgatcg ccctgaagga cgagaccaac cccatcgagg agtggcagca
     2701 catcgagcag ctgaagggca gcgccgaggg cagcatcaga taccccgcct ggaacggcat
     2761 catcagccag gaggtgctgg actacctgag cagctacatc aacaggcgga tctacccata
     2821 cgatgttcca gattacgctt aagaattcga tatcaagctt aataaaagat ctttattttc
     2881 attagatctg tgtgttggtt ttttgtgtcg gaccgagcgg ccgcaggaac ccctagtgat
     2941 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     3001 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     3061 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3121 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3181 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     3241 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3301 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     3361 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3421 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     3481 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     3541 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     3601 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     3661 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3721 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3781 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     3841 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3901 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3961 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4021 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4081 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4141 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4201 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     4261 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     4321 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     4381 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     4441 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     4501 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     4561 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     4621 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     4681 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     4741 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     4801 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     4861 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4921 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4981 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5041 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5101 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5161 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5221 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5281 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5341 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5401 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5461 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5521 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5581 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5641 tgctggcctt ttgctcacat gt
//
