LOCUS       sequence-387499-        7994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express ChRWR/Venus 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 7994)
  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 7994)
  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-EF1-alpha-Flex/3'USS-ChRWR/Venus(ATG mut)
FEATURES             Location/Qualifiers
     source          1..7994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        126..1304
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          357..1295
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1252..1272
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1310..1343)
                     /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(1394..1427)
                     /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(1446..2159,2160..2162,2163..2165))
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1491..1512)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1840..1859
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2099..2120
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2162..2171
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3242..3251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3286..3319
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3345..4049
                     /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    4070..4103
                     /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    4119..4707
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4172..4192)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4590..4601)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4719..5195
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4918..4937)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5235..5375
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5235..5364
                     /label=AAV2 ITR
     rep_origin      5450..5905
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5537..5556)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5747..5768
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5922..5941)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6041..6063
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6101..6119)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6187..6291
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6292..6360,6361..7152)
                     /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(6510..6529)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7323..7911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7812..7831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7973..7994,1..108)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
       61 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc
      121 cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
      181 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
      241 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
      301 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
      361 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
      421 gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg
      481 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
      541 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg
      601 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt
      661 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
      721 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc
      781 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
      841 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg
      901 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat
      961 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct
     1021 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc
     1081 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
     1141 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga
     1201 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc
     1261 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaaattca taacttcgta
     1321 tagcatacat tatacgaagt tatgcagaat ggtagctgga ttgtagctgc tattagcaat
     1381 atgaaacctc ttaataactt cgtataggat actttatacg aagttatgga tcctctagag
     1441 tcgacttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
     1501 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctggtagctc
     1561 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     1621 tggtcggcga gctgcacgcc gccgtcctcg atgttgtggc ggatcttgaa gttggccttg
     1681 atgccgttct tctgcttgtc ggcggtgata tagacgttgt ggctgttgta gttgtactcc
     1741 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1801 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1861 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     1921 tgctgcttca tgtggtcggg gtagcgggcg aagcactgca ggccgtagcc cagggtggtc
     1981 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatcagctt cagggtcagc
     2041 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     2101 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     2161 accatggtgg cgaccggtgg atccttgccg gtgcccttgt tgaccgcgcc agcctcggcc
     2221 tcgtcctcca ccagcgtctc gacctcaatc tcagtgccac caatgttcaa tttggtggtc
     2281 ttgcgaatgt cgccgtggat gaggatatgc tcgtggatca gcacgcgcag gtagtggccg
     2341 agcagacccc agcagttctt cgacatcagg tcaatgatgg tgtggccgac ggtggagccg
     2401 tacacgctca ggacgccgaa gccctcgggg ccgaggatga acaggatggg gaacataccc
     2461 catgatacga agaagagcca agccatgcca gtcaccacct ggcgacaccg gcccttcggc
     2521 acggtgtggt acgcctcaat gtagaccttg gctgcgttga agaatgtgta gatgccgtag
     2581 cacaggccca tcaggaagaa aatgacacgg acgtatccct tggacagcgc ggccgtggtg
     2641 ccccacacga tcgtgccgat atctgacacc agcagaccca tggtacgctt gttatagtcg
     2701 ttcgccagac ccgtaaggtt gctcagatgg ataagaatga cagggcaggt cagcagccac
     2761 tccgcgtaac gaagccacac ggtcttgttg ccgttggatg agtagatcac cgcaggttcg
     2821 tcaaactcat ggaaatactc gatgatgaac ttgatcatct cgatcgtggc cacgtaaatc
     2881 tcctcccagc cgcaagtaga cttccaggtc tggtagccgt agaacatcag gcagagcgct
     2941 gaaagcgcaa aagtaatcca ctgcagaatg ttggcagcca acttctcggc atttgttccg
     3001 ttggatttaa gccaagccaa gcagaagcac tggccgttgt tcgggatgca aataacagaa
     3061 ccattgttct caagagtgta tgaggtttgg aagagcatgc gctcgtgggc acggtggaaa
     3121 acgtagtcgg ggccatcctg agtcgcgacc ggcaccgtcg cgtcactgcc agtcgaggct
     3181 cctgcgctgc cggccgccag cgccactgcc agcgctaggg caagaagcca tggcctccgc
     3241 gccatggtgg caagcttgcc tcgagcagcg ctgctcgaga gatctataac ttcgtataat
     3301 gtatgctata cgaagttatc ctagcgctac cggtcgccac ctaggtgggt gaggatagcg
     3361 tgctgatcac cgagaacatg cacatgaaac tgtacatgga gggcaccgtg aacgaccacc
     3421 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgaaga
     3481 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct accagcttct
     3541 agtacggcag caaaaccttt atcaaccaca cccagggcat ccccgacttc tttaagcagt
     3601 ccttccctga gggcttcaca tgggagagga tcaccacata cgaagacggg ggcgtgctga
     3661 ccgctaccca ggacaccagc ctccagaacg gctgcctcat ctacaacgtc aagatcaacg
     3721 gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcca
     3781 gcaccgagat gctgtacccc gctgacagcg gcctgagagg ccatagccag atggccctga
     3841 agctcgtggg cgggggctac ctgcactgct ccctcaagac cacatacaga tccaagaaac
     3901 ccgctaagaa cctcaagatg cccggcttct acttcgtgga caggagactg gaaagaatca
     3961 aggaggccga caaagagacc tacgtcgagc agcacgagat ggctgtggcc aggtactgcg
     4021 acctgcctag caaactgggg cacagctgat gcggccgcga cgctagcgca taacttcgta
     4081 taaagtatcc tatacgaagt tatgcggccg catcgattaa tcaacctctg gattacaaaa
     4141 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     4201 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     4261 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     4321 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     4381 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     4441 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     4501 tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc
     4561 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     4621 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     4681 ggatctccct ttgggccgcc tccccgcatc tgatctacgg gtggcatccc tgtgacccct
     4741 ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa
     4801 taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga
     4861 ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct
     4921 attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct
     4981 gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga
     5041 ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc
     5101 tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat
     5161 tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg
     5221 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     5281 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     5341 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     5401 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     5461 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5521 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5581 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5641 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     5701 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5761 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     5821 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5881 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     5941 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     6001 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     6061 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     6121 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     6181 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6241 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     6301 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6361 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     6421 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     6481 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     6541 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     6601 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     6661 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6721 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6781 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6841 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6901 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6961 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     7021 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     7081 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     7141 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     7201 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7261 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7321 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7381 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7441 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     7501 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7561 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7621 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7681 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7741 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7801 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7861 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7921 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg
     7981 cagctgcgcg ctcg
//
