LOCUS       sequence-124532-        6180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to produce AAV expressing the FlpO recombinase and pig gRNA 
            towards 3 tumor suppressors: PTEN, p53 and SMAD4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6180)
  AUTHORS   Berthelsen MF, Riedel M, Cai H, Skaarup SH, Alstrup AKO, 
            Dagnaes-Hansen F, Luo Y, Jensen UB, Hager H, Liu Y, Callesen H, 
            Vendelbo MH, Jakobsen JE, Thomsen MK
  TITLE     The CRISPR/Cas9 Minipig-A Transgenic Minipig to Produce Specific 
            Mutations in Designated Tissues.
  JOURNAL   Cancers (Basel). 2021 Jun 16;13(12). pii: cancers13123024. doi: 
            10.3390/cancers13123024.
  PUBMED    34208747
REFERENCE   2  (bases 1 to 6180)
  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: "Cancers 
            (Basel)."; date: "2021-06-16"; volume: "13(12). pii: 
            cancers13123024. doi"; pages: " 10.3390/cancers13123024"
COMMENT     Sequence Label: AAV_3xgRNA;PTENA, p53B,SMAD4A_CAG_FlPO_synthPA
FEATURES             Location/Qualifiers
     source          1..6180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        153..401
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        422..497
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        510..758
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        779..854
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        867..1115
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1136..1211
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1224..1509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1511..1788
                     /label=chicken beta-actin promoter
     intron          1789..2017
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2029..2038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2035..2040,2041..2061,2062..3330)
                     /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             3331..3357
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     repeat_region   3443..3583
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3443..3572
                     /label=AAV2 ITR
     rep_origin      3658..4113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3745..3764)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3955..3976
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4130..4149)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4249..4271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4309..4327)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4395..4499
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4500..4568,4569..5360)
                     /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(4718..4737)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5531..6119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6020..6039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgcc gagagggcct atttcccatg attccttcat
      181 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
      241 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
      301 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
      361 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgcagcaatt cactgtaaag
      421 cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      481 tggcaccgag tcggtgcttt tttaccggtg agggcctatt tcccatgatt ccttcatatt
      541 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
      601 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      661 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      721 tcttggcttt atatatcttg tggaaaggac gaaacaccgt gcgtgtttgt gcctgtccgt
      781 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      841 caccgagtcg gtgctttttt atcgatgagg gcctatttcc catgattcct tcatatttgc
      901 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
      961 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     1021 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     1081 tggctttata tatcttgtgg aaaggacgaa acaccgaagg agaaaaaaga tgaatgtttt
     1141 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     1201 cgagtcggtg cttttttggt acccgttaca taacttacgg taaatggccc gcctggctga
     1261 ccgcccaacg acccccgccc attgacgtca atagtaacgc caatagggac tttccattga
     1321 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     1381 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     1441 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     1501 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     1561 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     1621 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
     1681 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     1741 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     1801 acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     1861 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     1921 ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa
     1981 ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggct
     2041 cctaagaaga agaggaaggt gatgagccag ttcgacatcc tgtgcaagac cccccccaag
     2101 gtgctggtgc ggcagttcgt ggagagattc gagaggccca gcggcgagaa gatcgccagc
     2161 tgtgccgccg agctgaccta cctgtgctgg atgatcaccc acaacggcac cgccatcaag
     2221 agggccacct tcatgagcta caacaccatc atcagcaaca gcctgagctt cgacatcgtg
     2281 aacaagagcc tgcagttcaa gtacaagacc cagaaggcca ccatcctgga ggccagcctg
     2341 aagaagctga tccccgcctg ggagttcacc atcatccctt acaacggcca gaagcaccag
     2401 agcgacatca ccgacatcgt gtccagcctg cagctgcagt tcgagagcag cgaggaggcc
     2461 gacaagggca acagccacag caagaagatg ctgaaggccc tgctgtccga gggcgagagc
     2521 atctgggaga tcaccgagaa gatcctgaac agcttcgagt acaccagcag gttcaccaag
     2581 accaagaccc tgtaccagtt cctgttcctg gccacattca tcaactgcgg caggttcagc
     2641 gacatcaaga acgtggaccc caagagcttc aagctggtgc agaacaagta cctgggcgtg
     2701 atcattcagt gcctggtgac cgagaccaag acaagcgtgt ccaggcacat ctactttttc
     2761 agcgccagag gcaggatcga ccccctggtg tacctggacg agttcctgag gaacagcgag
     2821 cccgtgctga agagagtgaa caggaccggc aacagcagca gcaacaagca ggagtaccag
     2881 ctgctgaagg acaacctggt gcgcagctac aacaaggccc tgaagaagaa cgccccctac
     2941 cccatcttcg ctatcaagaa cggccctaag agccacatcg gcaggcacct gatgaccagc
     3001 tttctgagca tgaagggcct gaccgagctg acaaacgtgg tgggcaactg gagcgacaag
     3061 agggcctccg ccgtggccag gaccacctac acccaccaga tcaccgccat ccccgaccac
     3121 tacttcgccc tggtgtccag gtactacgcc tacgacccca tcagcaagga gatgatcgcc
     3181 ctgaaggacg agaccaaccc catcgaggag tggcagcaca tcgagcagct gaagggcagc
     3241 gccgagggca gcatcagata ccccgcctgg aacggcatca tcagccagga ggtgctggac
     3301 tacctgagca gctacatcaa caggcggatc tacccatacg atgttccaga ttacgcttaa
     3361 gaattcgata tcaagcttaa taaaagatct ttattttcat tagatctgtg tgttggtttt
     3421 ttgtgtcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     3481 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     3541 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     3601 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     3661 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3721 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3781 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3841 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     3901 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3961 tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag
     4021 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4081 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4141 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4201 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4261 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4321 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4381 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4441 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4501 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4561 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4621 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4681 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4741 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4801 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4861 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4921 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4981 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5041 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5101 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5161 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5221 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5281 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5341 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5401 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5461 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5521 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5581 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5641 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     5701 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5761 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5821 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5881 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5941 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6001 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6061 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6121 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
