LOCUS       sequence-387491-        7249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express GFP in the 
            presence of Cre including the shortage of a unilateral spacer 
            sequence(USS).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7249)
  AUTHORS   Matsushita N, Kato S, Nishizawa K, Sugawara M, Takeuchi K, Miyasaka 
            Y, Mashimo T, Kobayashi K
  TITLE     Highly selective transgene expression through the flip-excision 
            switch system by using a unilateral spacer sequence.
  JOURNAL   Cell Rep Methods. 2023 Jan 18;3(2):100393. doi: 
            10.1016/j.crmeth.2022.100393. eCollection 2023 Feb 27.
  PUBMED    36936079
REFERENCE   2  (bases 1 to 7249)
  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: "Cell Rep 
            Methods."; date: "2023-01-18"; volume: "3(2):100393. doi"; pages: " 
            10.1016/j.crmeth.2022.100393. eCollection 2023 Feb 27"
COMMENT     Sequence Label: pAAV-CAGGS-Flex/3'USS(553)-GFP
FEATURES             Location/Qualifiers
     source          1..7249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        146..525
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        527..804
                     /label=chicken beta-actin promoter
     intron          805..1821
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1745..1767
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1829..1848
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1876..1909)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1960..1993)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             complement(join(2000..2713,2714..2716,2717..2719))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2003..2713,2714..2716,2717..2719))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2045..2066)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2394..2413
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2653..2674
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2716..2725
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2760..2793
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3347..3380
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    3396..3984
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3449..3469)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3867..3878)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3996..4472
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4195..4214)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4512..4652
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4512..4641
                     /label=AAV2 ITR
     rep_origin      4727..5182
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4814..4833)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5024..5045
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5199..5218)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5318..5340
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5378..5396)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5464..5568
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5569..5637,5638..6429)
                     /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(5787..5806)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6600..7188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7089..7108
                     /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 gcgtcgacat tgattattga ctagttatta atagtaatca
      181 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      241 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      301 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      361 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      421 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      481 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc
      541 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
      601 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccgggs
      661 ggggsggggg ggggsggggg gsggggsggg gsgrggcgga gaggtgcggc ggcagccaat
      721 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
      781 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
      841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
      901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
      961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
     1021 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
     1081 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt
     1141 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
     1201 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
     1261 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
     1321 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
     1381 caggtggggg tgccgggcgg ggcggggccg cckcgggccg gggggggctc gggggrgggg
     1441 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
     1501 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
     1561 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
     1621 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     1681 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     1741 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     1801 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     1861 tttggcaaag aattcataac ttcgtatagc atacattata cgaagttatg cagaatggta
     1921 gctggattgt agctgctatt agcaatatga aacctcttaa taacttcgta taggatactt
     1981 tatacgaagt tatggatcct tacttgtaca gctcgtccat gccgagagtg atcccggcgg
     2041 cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg
     2101 cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
     2161 tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct
     2221 tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt
     2281 tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct
     2341 tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt
     2401 agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg
     2461 acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt
     2521 aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga
     2581 acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact
     2641 tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct
     2701 cctcgccctt gctcaccatg gtggcaagct tgcctcgagc agcgctgctc gagagatcta
     2761 taacttcgta taatgtatgc tatacgaagt tattgcacat ccgagggcga aggcaagccc
     2821 tacgagggca cccagaccat gaagatcaag gtggtcgagg gcggccctct ccccttcgcc
     2881 ttcgacatcc tggctaccag cttcatgtac ggcagcaaaa cctttatcaa ccacacccag
     2941 ggcatccccg acttctttaa gcagtccttc cctgagggct tcacatggga gaggatcacc
     3001 acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca gaacggctgc
     3061 ctcatctaca acgtcaagat caacggggtg aacttcccat ccaacggccc tgtgatgcag
     3121 aagaaaacac tcggctggga ggccagcacc gagatgctgt accccgctga cagcggcctg
     3181 agaggccata gccagatggc cctgaagctc gtgggcgggg gctacctgca ctgctccctc
     3241 aagaccacat acagatccaa gaaacccgct aagaacctca agatgcccgg cttctacttc
     3301 gtggacagga gactggaaag aatcaaggag gccgacaaag agacctataa cttcgtataa
     3361 agtatcctat acgaagttat gcggccgcat cgattaatca acctctggat tacaaaattt
     3421 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     3481 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     3541 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     3601 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     3661 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     3721 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     3781 tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc
     3841 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     3901 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     3961 tctccctttg ggccgcctcc ccgcatctga tctacgggtg gcatccctgt gacccctccc
     4021 cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa
     4081 aattaagttg catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg
     4141 gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt
     4201 gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg
     4261 ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca
     4321 ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg
     4381 tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac
     4441 aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac
     4501 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4561 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     4621 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     4681 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     4741 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4801 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4861 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4921 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     4981 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5041 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     5101 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5161 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     5221 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5281 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5341 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     5401 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5461 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5521 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5581 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5641 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5701 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5761 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5821 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5881 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5941 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     6001 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     6061 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     6121 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6181 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6241 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     6301 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6361 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6421 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6481 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6541 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6601 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6661 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6721 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6781 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6841 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6901 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6961 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7021 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7081 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7141 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7201 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
