LOCUS       sequence-387492-        7502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express Turbo/GFP in the
            presence of Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7502)
  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 7502)
  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-Turbo/GFP
FEATURES             Location/Qualifiers
     source          1..7502
                     /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)."
     regulatory      2016..2025
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2022..2729
                     /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="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHSQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     CDS             complement(join(2756..3469,3470..3472,3473..3475))
                     /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(2759..3469,3470..3472,3473..3475))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2801..2822)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3150..3169
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3409..3430
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3472..3481
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3516..3549
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3600..3633
                     /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    3649..4237
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3702..3722)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4120..4131)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4249..4725
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4448..4467)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4765..4905
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4765..4894
                     /label=AAV2 ITR
     rep_origin      4980..5435
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5067..5086)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5277..5298
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5452..5471)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5571..5593
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5631..5649)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5717..5821
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5822..5890,5891..6682)
                     /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(6040..6059)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6853..7441
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7342..7361
                     /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 gggggggggs ggggsgrggg 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 tatggatccc ctagcgctac cggtcgccac catggtgggt gaggatagcg
     2041 tgctgatcac cgagaacatg cacatgaaac tgtacatgga gggcaccgtg aacgaccacc
     2101 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgaaga
     2161 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca
     2221 tgtacggcag caaaaccttt atcaaccaca cccagggcat ccccgacttc tttaagcagt
     2281 ccttccctga gggcttcaca tgggagagga tcaccacata cgaagacggg ggcgtgctga
     2341 ccgctaccca ggacaccagc ctccagaacg gctgcctcat ctacaacgtc aagatcaacg
     2401 gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcca
     2461 gcaccgagat gctgtacccc gctgacagcg gcctgagagg ccatagccag atggccctga
     2521 agctcgtggg cgggggctac ctgcactgct ccctcaagac cacatacaga tccaagaaac
     2581 ccgctaagaa cctcaagatg cccggcttct acttcgtgga caggagactg gaaagaatca
     2641 aggaggccga caaagagacc tacgtcgagc agcacgagat ggctgtggcc aggtactgcg
     2701 acctgcctag caaactgggg cacagctgat gcggccgcga cgctagcgca gatccttact
     2761 tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca
     2821 tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt
     2881 tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga
     2941 gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct
     3001 tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc
     3061 ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg
     3121 tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga
     3181 tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca
     3241 tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc acgagggtgg
     3301 gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg
     3361 tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca
     3421 gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc accatggtgg
     3481 caagcttgcc tcgagcagcg ctgctcgaga gatctataac ttcgtataat gtatgctata
     3541 cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa
     3601 taacttcgta taaagtatcc tatacgaagt tatgcggccg catcgattaa tcaacctctg
     3661 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3721 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3781 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3841 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3901 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3961 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     4021 aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc
     4081 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     4141 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     4201 cagacgagtc ggatctccct ttgggccgcc tccccgcatc tgatctacgg gtggcatccc
     4261 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     4321 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     4381 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     4441 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     4501 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     4561 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     4621 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     4681 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
     4741 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     4801 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4861 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4921 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4981 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5041 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5101 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5161 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     5221 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5281 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     5341 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5401 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     5461 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     5521 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5581 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     5641 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     5701 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5761 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5821 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5881 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5941 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6001 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6061 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6121 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6181 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6241 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6301 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6361 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6421 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6481 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6541 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6601 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6661 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6721 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6781 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6841 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6901 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6961 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     7021 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7081 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7141 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7201 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7261 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7321 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7381 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7441 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7501 gt
//
