LOCUS       sequence-383031-        6981 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 6981)
  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 6981)
  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(285)-GFP
FEATURES             Location/Qualifiers
     source          1..6981
                     /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)."
     regulatory      2804..2813
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3079..3112
                     /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    3128..3716
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3181..3201)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3599..3610)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3728..4204
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3927..3946)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4244..4384
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4244..4373
                     /label=AAV2 ITR
     rep_origin      4459..4914
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4546..4565)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4756..4777
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4931..4950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5050..5072
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5110..5128)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5196..5300
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5301..5369,5370..6161)
                     /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(5519..5538)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6332..6920
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6821..6840
                     /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 cgcgccaggc
      661 ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 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 cctcgggccg gggagggctc gggggagggg
     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 tatgctaccg gtcgccacca tggtgggtga
     2821 ggatagcgtg ctgatcaccg agaacatgca catgaaactg tacatggagg gcaccgtgaa
     2881 cgaccaccac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     2941 catgaagatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     3001 cagcttcatg tacggcagca aaacctttat caaccacacc cagggcatcc ccgacttctt
     3061 taagcagtcc ttccctgaat aacttcgtat aaagtatcct atacgaagtt atgcggccgc
     3121 atcgattaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3181 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3241 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3301 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3361 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3421 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3481 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     3541 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3601 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3661 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatct
     3721 gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc
     3781 cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
     3841 ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg
     3901 gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca
     3961 atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc
     4021 cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta
     4081 gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta
     4141 cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt
     4201 ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     4261 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4321 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4381 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4441 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4501 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     4561 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4621 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     4681 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4741 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     4801 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     4861 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     4921 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     4981 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5041 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5101 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5161 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5221 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5281 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5341 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5401 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5461 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5521 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5581 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5641 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5701 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5761 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5821 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5881 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5941 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6001 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6061 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6121 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6181 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6241 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6301 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6361 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6421 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6481 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6541 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6601 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6661 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6721 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6781 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6841 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6901 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6961 gctggccttt tgctcacatg t
//
