LOCUS       sequence-387494-        7446 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express GFP in the 
            presence of Cre and the leakage expression is significantly reduced 
            without Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7446)
  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 7446)
  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-GFP(ATG mut)
FEATURES             Location/Qualifiers
     source          1..7446
                     /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"
     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)."
     CDS             2819..3523
                     /codon_start=1
                     /product="far-red-shifted derivative of red fluorescent 
                     protein from Entacmaea quadricolor, also known as Katushka 
                     (Shcherbo et al., 2007)"
                     /label=TurboFP635
                     /note="mammalian codon-optimized"
                     /translation="VGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTMK
                     IKVVEGGPLPFAFDILATSF*YGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGGV
                     LTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHSQM
                     ALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAVA
                     RYCDLPSKLGHS"
     protein_bind    3544..3577
                     /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    3593..4181
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3646..3666)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4064..4075)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4193..4669
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4392..4411)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4709..4849
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4709..4838
                     /label=AAV2 ITR
     rep_origin      4924..5379
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5011..5030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5221..5242
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5396..5415)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5515..5537
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5575..5593)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5661..5765
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5766..5834,5835..6626)
                     /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(5984..6003)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6797..7385
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7286..7305
                     /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 cgcgccgggg
      661 ggggsggggg ggggsggggg gsggggsggg gsgrggcggr gaggkgcggc 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 ccycgggccg 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 tatcctagcg ctaccggtcg ccacctaggt
     2821 gggtgaggat agcgtgctga tcaccgagaa catgcacatg aaactgtaca tggagggcac
     2881 cgtgaacgac caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     2941 ccagaccatg aagatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     3001 ggctaccagc ttctagtacg gcagcaaaac ctttatcaac cacacccagg gcatccccga
     3061 cttctttaag cagtccttcc ctgagggctt cacatgggag aggatcacca catacgaaga
     3121 cgggggcgtg ctgaccgcta cccaggacac cagcctccag aacggctgcc tcatctacaa
     3181 cgtcaagatc aacggggtga acttcccatc caacggccct gtgatgcaga agaaaacact
     3241 cggctgggag gccagcaccg agatgctgta ccccgctgac agcggcctga gaggccatag
     3301 ccagatggcc ctgaagctcg tgggcggggg ctacctgcac tgctccctca agaccacata
     3361 cagatccaag aaacccgcta agaacctcaa gatgcccggc ttctacttcg tggacaggag
     3421 actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg agatggctgt
     3481 ggccaggtac tgcgacctgc ctagcaaact ggggcacagc tgatgcggcc gcgacgctag
     3541 cgcataactt cgtataaagt atcctatacg aagttatgcg gccgcatcga ttaatcaacc
     3601 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3661 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3721 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3781 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3841 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3901 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3961 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     4021 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     4081 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     4141 ccctcagacg agtcggatct ccctttgggc cgcctccccg catctgatct acgggtggca
     4201 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac
     4261 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa
     4321 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag
     4381 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc
     4441 aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt
     4501 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac
     4561 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc
     4621 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta
     4681 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc
     4741 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4801 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4861 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4921 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4981 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     5041 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     5101 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     5161 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5221 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     5281 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     5341 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     5401 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     5461 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     5521 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     5581 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     5641 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5701 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5761 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5821 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5881 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5941 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6001 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6061 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6121 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6181 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6241 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6301 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6361 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6421 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6481 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6541 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6601 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6661 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6721 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6781 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6841 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6901 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6961 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     7021 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     7081 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7141 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7201 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7261 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7321 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7381 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7441 acatgt
//
