LOCUS       sequence-256055-        8059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double floxed ChRmine-eYFP under the control of Ef1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8059)
  AUTHORS   Marshel JH, Kim YS, Machado TA, Quirin S, Benson B, Kadmon J, Raja 
            C, Chibukhchyan A, Ramakrishnan C, Inoue M, Shane JC, McKnight DJ, 
            Yoshizawa S, Kato HE, Ganguli S, Deisseroth K
  TITLE     Cortical layer-specific critical dynamics triggering perception.
  JOURNAL   Science. 2019 Aug 9;365(6453). pii: science.aaw5202. doi: 
            10.1126/science.aaw5202. Epub 2019 Jul 18.
  PUBMED    31320556
REFERENCE   2  (bases 1 to 8059)
  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: "Science."; 
            date: "2019-08-9"; volume: "365(6453). pii: science.aaw5202. doi"; 
            pages: " 10.1126/science.aaw5202. Epub 2019 Jul 18"
COMMENT     Sequence Label: pAAV-Ef1a-DIO ChRmine-eYFP-WPRE
FEATURES             Location/Qualifiers
     source          1..8059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      7..16
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1009..1011,1012..1722)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1051..1072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1312..1331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1659..1680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1758..1791
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1842..1875
                     /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)."
     promoter        complement(1906..3084)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1915..2853
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(1938..1958)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   3185..3314
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3376..3964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3456..3475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4135..4926,4927..4995))
                     /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     4758..4777
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4996..5100)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5168..5186
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5224..5246)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5346..5365
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5382..5837)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5519..5540)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5731..5750
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5912..6052
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5923..6052
                     /label=AAV2 ITR
     polyA_signal    6092..6299
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6288..6305
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     misc_feature    7331..7919
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             7437..7448
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7846..7866
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(7944..7977)
                     /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(join(8028..8059,1..2))
                     /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)."
ORIGIN
        1 atgctagcca ccatggcaca cgcaccaggc accgaccaga tgttctacgt gggcacaatg
       61 gacggctggt atctggatac caagctgaac tccgtggcca tcggcgccca ctggtcttgc
      121 tttatcgtgc tgacaatcac cacattctac ctgggctatg agtcctggac atctcggggc
      181 cctagcaaga gaacctcctt ttacgccggc tatcaggagg agcagaacct ggccctgttt
      241 gtgaatttct ttgccatgct gagctacttc ggcaagatcg tggccgacac actgggccac
      301 aacttcggcg atgtgggccc cttcatcatc ggcttcggca attacaggta tgccgactac
      361 atgctgacct gcccaatgct ggtgtacgat ctgctgtatc agctgagggc cccctatcgc
      421 gtgtcttgta gcgccatcat ctttgccatc ctgatgtctg gcgtgctggc cgagttctac
      481 gcagagggcg accctaggct gaggaatggc gcctacgcct ggtatggctt tggctgtttc
      541 tggtttatct tcgcctactc tatcgtgatg agcatcgtgg ccaagcagta tagccggctg
      601 gcccagctgg cccaggatac aggcgccgag cacagcctgc acgtgctgaa gttcgccgtg
      661 tttaccttct ccatgctgtg gattctgttt cccctggtgt gggccatctg ccctagaggc
      721 ttcggctgga tcgacgataa ctggacagag gtggcccact gcgtgtgcga catcgtggcc
      781 aagtcctgtt acggctttgc cctggcccgg ttcagaaaga cctatgatga ggagctgttt
      841 cggctgctgg agcagctggg acacgacgag gatgagttcc agaagctgga gctggatatg
      901 aggctgagca gcaatggcga gcgcctgcgg agactgtctg cggccgccaa gagcaggatc
      961 accagcgagg gcgagtacat ccccctggac cagatcgaca tcaacgtggt gagcaagggc
     1021 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1081 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1141 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
     1201 ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     1261 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1321 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1381 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1441 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1501 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1561 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag
     1621 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1681 accgccgccg ggatcactct cggcatggac gagctgtaca agttctgcta cgagaacgag
     1741 gtgtaatagg gcgcgccata acttcgtata atgtatgcta tacgaagtta ttaagaggtt
     1801 tcatattgct aatagcagct acaatccagc taccattctg cataacttcg tataaagtat
     1861 cctatacgaa gttattccgg agtcgactct agaggatccg gtacctcacg acacctgaaa
     1921 tggaagaaaa aaactttgaa ccactgtctg aggcttgaga atgaaccaag atccaaactc
     1981 aaaaagggca aattccaagg agaattacat caagtgccaa gctggcctaa cttcagtctc
     2041 cacccactca gtgtggggaa actccatcgc ataaaacccc tccccccaac ctaaagacga
     2101 cgtactccaa aagctcgaga actaatcgag gtgcctggac ggcgcccggt actccgtgga
     2161 gtcacatgaa gcgacggctg aggacggaaa ggcccttttc ctttgtgtgg gtgactcacc
     2221 cgcccgctct cccgagcgcc gcgtcctcca ttttgagctc cctgcagcag ggccgggaag
     2281 cggccatctt tccgctcacg caactggtgc cgaccgggcc agccttgccg cccagggcgg
     2341 ggcgatacac ggcggcgcga ggccaggcac cagagcaggc cggccagctt gagactaccc
     2401 ccgtccgatt ctcggtggcc gcgctcgcag gccccgcctc gccgaacatg tgcgctggga
     2461 cgcacgggcc ccgtcgccgc ccgcggcccc aaaaaccgaa ataccagtgt gcagatcttg
     2521 gcccgcattt acaagactat cttgccagaa aaaaagcgtc gcagcaggtc atcaaaaatt
     2581 ttaaatggct agagacttat cgaaagcagc gagacaggcg cgaaggtgcc accagattcg
     2641 cacgcggcgg ccccagcgcc caggccaggc ctcaactcaa gcacgaggcg aaggggctcc
     2701 ttaagcgcaa ggcctcgaac tctcccaccc acttccaacc cgaagctcgg gatcaagaat
     2761 cacgtactgc agccaggtgg aagtaattca aggcacgcaa gggccataac ccgtaaagag
     2821 gccaggcccg cgggaaccac acacggcact tacctgtgtt ctggcggcaa acccgttgcg
     2881 aaaaagaacg ttcacggcga ctactgcact tatatacggt tctcccccac cctcgggaaa
     2941 aaggcggagc cagtacacga catcactttc ccagtttacc ccgcgccacc ttctctaggc
     3001 accggttcaa ttgccgaccc ctccccccaa cttctcgggg actgtgggcg atgtgcgctc
     3061 tgcccactga cgggcaccgg agccaattcc cactcctttc aagacctaga aggtccatta
     3121 gctgcaaaga ttcctctctg tttaaaactt tatccatctt tgcaaagctt acgcgtgcgg
     3181 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     3241 gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg
     3301 cagctgcctg caggacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3361 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3421 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3481 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3541 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3601 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3661 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3721 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3781 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3841 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3901 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3961 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4021 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4081 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4141 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4201 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4261 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4321 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4381 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4441 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4501 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4561 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4621 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4681 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4741 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4801 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4861 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4921 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4981 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5041 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5101 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     5161 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     5221 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     5281 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     5341 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat
     5401 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aatagaccga
     5461 aatcggcaaa atcccttata aatcaaaaga atagcccgag atagagttga gtgttgttcc
     5521 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     5581 cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc
     5641 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     5701 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa
     5761 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     5821 gccgctacag ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat accgcacaga
     5881 tgcgtaagga gaaaataccg catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc
     5941 actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     6001 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
     6061 tcggtccgca cgtggttacc tacaaaatca gtccccagca tgcctgctat tgtcttccca
     6121 atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag
     6181 acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag
     6241 ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt
     6301 cgaggcttat tttttttttt tttttttcaa acaataactt ttattttata cttctctata
     6361 ctttgtagca aatctttttt tgctgaattt aatttataat aaacttttta aattacatct
     6421 ctctctcttt tttttttaaa atcaaggctc ttttatgtca aaatcttttt ttagctatat
     6481 tttagattaa catttaacat cccccccttg tgatctatac cgttggatat tcaggtatta
     6541 ctgtgtgtgt aacagctaaa acaagaggga ggagggaaaa taaaggcagt gaacttggac
     6601 ggatgcatca acaacagcag ataaagctaa cccctcagtg accatagcag catgtcttct
     6661 ggaagccttt actcttaccc cagagatttc ctcagcccct tccctctctc cctcctatcc
     6721 tccaaacaca aagccaacag tctgtccttt cgcttttctt gaggagaaat gtgcagtgga
     6781 aatgatcaaa acaagatact gtggaagaac gacgtgagcg tgaattattc accgtatgtt
     6841 tcgtccgtgg acatctctct tgaattcatt cccctggcct tctcctctcc tcgccttcct
     6901 attaggagga gcccatcatt tttcatgtta ctagcatgat taatatagta ggtggcccag
     6961 ggccgttcct gagattcttt tgccaacaaa aaaaattaat aataataaaa ttaaaaaatg
     7021 gggtgggttt gccgttttct tatttgtgtg tgaagacgga ctaaagctga ggtccgattt
     7081 tggctgtgct tgcttgctca ctgattggta acatttggca aataaaacac aagaaatcaa
     7141 acgaaataaa aaagagaatc aggatttccc acaatcctat atgagtgttt tcagcatcac
     7201 agagaatcac aacgtcccca aagaactaat ggatcttcct ctcgatttcc gggagcatgg
     7261 agtgagtgtg ggtgggcgcg cggggggagc tggccccacg cagatctctc gagcagcgct
     7321 cggtatcgat gcggggaggc ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga
     7381 agacgcggaa gaggccgcag agccggcagc aggccgcggg aaggaaggtc cgctggattg
     7441 agggccgaag ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc aacataggcg
     7501 agcagccaag gaaaggacga tgatttcccc gacaacacca cggaattgtc agtgcccaac
     7561 agccgagccc ctgtccagca gcgggcaagg caggcggcga tgagttccgc cgtggcaata
     7621 gggaggggga aagcgaaagt cccggaaagg agctgacagg tggtggcaat gccccaacca
     7681 gtgggggttg cgtcagcaaa cacagtgcac accacgccac gttgcctgac aacgggccac
     7741 aactcctcat aaagagacag caaccaggat ttatacaagg aggagaaaat gaaagccata
     7801 cgggaagcaa tagcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga
     7861 gcaacatagt taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgatta
     7921 tcgataagct tgatatcgaa ttcataactt cgtatagcat acattatacg aagttattct
     7981 ttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaaata acttcgtata
     8041 ggatacttta tacgaagtt
//
