LOCUS       sequence-387498-        6906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express mCherry 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 6906)
  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 6906)
  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-mCherry(ATG full mut)
FEATURES             Location/Qualifiers
     source          1..6906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        148..1326
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          379..1317
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1274..1294
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1333..1366)
                     /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(1417..1450)
                     /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(1469..2179)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1747..1766)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2016..2034
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2176..2185
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2220..2253
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2279..2983
                     /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="VGEDSVLITEN*H*KLY*EGTVNDHHFKCTSEGEGKPYEGTQT*K
                     IKVVEGGPLPFAFDILATSF*YGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGGV
                     LTATQDTSLQNGCLIYNVKINGVNFPSNGPV*QKKTLGWEASTE*LYPADSGLRGHSQ*
                     ALKLVGGGYLHCSLKTTYRSKKPAKNLK*PGFYFVDRRLERIKEADKETYVEQHE*AVA
                     RYCDLPSKLGHS"
     protein_bind    3004..3037
                     /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    3053..3641
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3106..3126)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3524..3535)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3653..4129
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3852..3871)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4169..4309
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4169..4298
                     /label=AAV2 ITR
     rep_origin      4384..4839
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4471..4490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4681..4702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4856..4875)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4975..4997
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5035..5053)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5121..5225
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5226..5294,5295..6086)
                     /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(5444..5463)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6257..6845
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6746..6765
                     /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 gccgtgaggc tccggtgccc gtcagtgggc agagcgcaca
      181 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
      241 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
      301 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
      361 tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg
      421 ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat
      481 cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct
      541 tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg
      601 tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga
      661 tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg
      721 cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc
      781 acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct
      841 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg
      901 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc
      961 cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca
     1021 cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag
     1081 taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta
     1141 ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa
     1201 gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga
     1261 tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg
     1321 tcgtgagaat tcataacttc gtatagcata cattatacga agttatgcag aatggtagct
     1381 ggattgtagc tgctattagc aatatgaaac ctcttaataa cttcgtatag gatactttat
     1441 acgaagttat ggatcctcta gagtcgactt acttgtacag ctcgtccatg ccgccggtgg
     1501 agtggcggcc ctcggcgcgt tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg
     1561 tgatgtccaa cttgatgttg acgttgtagg cgccgggcag ctgcacgggc ttcttggcct
     1621 tgtaggtggt cttgacctca gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct
     1681 tgatctcgcc cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc
     1741 ccatggtctt cttctgcatt acggggccgt cggaggggaa gttggtgccg cgcagcttca
     1801 ccttgtagat gaactcgccg tcctgcaggg aggagtcctg ggtcacggtc accacgccgc
     1861 cgtcctcgaa gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcaagt
     1921 agtcggggat gtcggcgggg tgcttcacgt aggccttgga gccgtacatg aactgagggg
     1981 acaggatgtc ccaggcgaag ggcagggggc cacccttggt caccttcagc ttggcggtct
     2041 gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca
     2101 cggagccctc catgtgcacc ttgaagcgca tgaactcctt gatgatggcc atgttatcct
     2161 cctcgccctt gctcaccatg gtggcaagct tgcctcgagc agcgctgctc gagagatcta
     2221 taacttcgta taatgtatgc tatacgaagt tatcctagcg ctaccggtcg ccacctaggt
     2281 gggtgaggat agcgtgctga tcaccgagaa ctagcactag aaactgtact aggagggcac
     2341 cgtgaacgac caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     2401 ccagacctag aagatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     2461 ggctaccagc ttctagtacg gcagcaaaac ctttatcaac cacacccagg gcatccccga
     2521 cttctttaag cagtccttcc ctgagggctt cacatgggag aggatcacca catacgaaga
     2581 cgggggcgtg ctgaccgcta cccaggacac cagcctccag aacggctgcc tcatctacaa
     2641 cgtcaagatc aacggggtga acttcccatc caacggccct gtgtagcaga agaaaacact
     2701 cggctgggag gccagcaccg agtagctgta ccccgctgac agcggcctga gaggccatag
     2761 ccagtaggcc ctgaagctcg tgggcggggg ctacctgcac tgctccctca agaccacata
     2821 cagatccaag aaacccgcta agaacctcaa gtagcccggc ttctacttcg tggacaggag
     2881 actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg agtaggctgt
     2941 ggccaggtac tgcgacctgc ctagcaaact ggggcacagc tgatgcggcc gcgacgctag
     3001 cgcataactt cgtataaagt atcctatacg aagttatgcg gccgcatcga ttaatcaacc
     3061 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3121 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3181 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3241 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3301 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3361 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3421 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     3481 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3541 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3601 ccctcagacg agtcggatct ccctttgggc cgcctccccg catctgatct acgggtggca
     3661 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac
     3721 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa
     3781 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag
     3841 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc
     3901 aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt
     3961 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac
     4021 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc
     4081 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta
     4141 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc
     4201 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4261 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4321 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4381 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4441 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4501 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4561 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     4621 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4681 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     4741 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     4801 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     4861 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4921 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4981 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     5041 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     5101 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5161 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5221 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5281 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5341 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5401 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5461 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5521 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5581 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5641 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5701 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5761 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5821 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5881 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5941 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6001 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6061 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6121 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6181 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6241 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6301 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6361 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6421 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6481 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6541 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6601 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6661 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6721 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6781 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6841 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6901 acatgt
//
