LOCUS       sequence-323834-        6338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of ChromeT-GFP under the Syn promoter, in 
            floxed/reversed (Cre-dependent) manner..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6338)
  AUTHORS   Cho YK, Park D, Yang A, Chen F, Chuong AS, Klapoetke NC, Boyden ES
  TITLE     Multidimensional screening yields channelrhodopsin variants having 
            improved photocurrent and order-of-magnitude reductions in calcium 
            and proton currents.
  JOURNAL   J Biol Chem. 2019 Jan 4. pii: RA118.006996. doi: 
            10.1074/jbc.RA118.006996.
  PUBMED    30610117
REFERENCE   2  (bases 1 to 6338)
  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: "J Biol 
            Chem. 2019 Jan 4. pii: RA118.006996. doi: 10.1074/jbc.RA118.006996."
COMMENT     Sequence Label: pAAV-Syn-FLEX-rc [ChromeT-GFP]
FEATURES             Location/Qualifiers
     source          1..6338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     promoter        236..683
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    699..732
                     /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)."
     protein_bind    795..828
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(842..1555,1556..1558))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(845..1555,1556..1558))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(887..908)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1236..1255
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1495..1516
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1580..2506)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYSYQTWKSTCGWEEIYVCAIAMVKVILEFFFEFKN
                     PSMLYLATGGRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      2503..2512
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2523..2556)
                     /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)."
     protein_bind    complement(2627..2660)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2693..3281
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2746..2766)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3164..3175)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3322..3529
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3699..3828
                     /label=AAV2 ITR
     rep_origin      complement(4065..4653)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4145..4164)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4824..5615,5616..5684))
                     /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     5447..5466
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5685..5789)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5815..6270)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5952..5973)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6164..6183
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
      181 tctaggaaga tcgtaggtta attaagtgtc tagactgcag agggccctgc gtatgagtgc
      241 aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg
      301 acccactgga caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg
      361 ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag
      421 tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga
      481 cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg
      541 ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc
      601 atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag
      661 tcgtgtcgtg cctgagagcg cagtcgagaa gctagcccat aacttcgtat aaagtatcct
      721 atacgaagtt atatcaaaat aggaagacca atgcttcacc atcgacccga attgccaagc
      781 atcaccatcg acccataact tcgtataatg tatgctatac gaagttatac tagaattctc
      841 attacttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca
      901 ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt
      961 agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt
     1021 cggcgagctg cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc
     1081 cgttcttctg cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct
     1141 tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca
     1201 ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga
     1261 agaagatggt gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct
     1321 gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga
     1381 gggtgggcca gggcacgggc agcttgccgg tggtgcaaat gaacttcagg gtcagcttgc
     1441 cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc
     1501 cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcactg
     1561 ctactaccgg tgccgctggc acggctccgg cctcggcttc gtcttcgacg agagtctcga
     1621 cctcgatctc cgttccgccg atgttcagtt tggtggtttt gcggatatct ccgtgaatca
     1681 atatgtgctc gtggatcagg acgcgcaggt agtgtcccaa caacccccaa caatttttac
     1741 tcatcagatc aataatcgtg tgacctacgg tggagccata gacgctcagg acgccaaaac
     1801 cttcgggccc caaaatgaag agaattggga acatacccca gctcacgaaa aacagccatg
     1861 ccatgccggt cacgacctgg cggcaccgac cctttggcac agtatgataa ccctcgatat
     1921 atgctttggc ggcgtgaaaa aatgtgttcg cgccatagca caatccaaga caaaagaaga
     1981 tgactttaac atagccggtt gccatggcgc tggtagcccc ccacacgata gtcccgatgt
     2041 ctgagacaag gagtcccatg gttctcctgc tgtagtcgtt gctcaggccg gtgaggttgc
     2101 tcaggtggat aaggatgaca ggacaagtga gcagccactc tgcatagcgc agccactgca
     2161 cccgacctcc tgtggcaagg tagagcatag agggattctt aaactcaaaa aagaactcga
     2221 gaatcacctt aaccattgca atggcgcaca catagatctc ctcccagccg catgtagatt
     2281 tccaggtttg gtaagaatag aacatcagca gcaaaatgct gaatcctgct gcaagccact
     2341 gcaggacatt tgacgcggtc tgagcgccgt tcgtgccgcg agattcaatc catccggcac
     2401 agtaacattg atcctcaggg accaggacgg acccgttcac caccacagga ttagtaacga
     2461 acaaaagttc gcgtccgacg gcagacaaag cgccgccata gtccatggtg gcggatccta
     2521 gtataacttc gtataggata ctttatacga agttatcatt gggattcttc ctattttgat
     2581 ccaagcatca ccatcgaccc tctagtccag atctcaccat cgacccataa cttcgtatag
     2641 catacattat acgaagttat gtccctcgaa gaggttcgcg aagcttatcg ataatcaacc
     2701 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     2761 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     2821 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     2881 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     2941 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3001 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3061 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     3121 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3181 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3241 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacccgg
     3301 gcggccgctc ataaagctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3361 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3421 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3481 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtccgga
     3541 ctgtacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa
     3601 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atcttcctag agcatggcta
     3661 cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt
     3721 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3781 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcattaatga
     3841 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3901 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3961 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4021 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4081 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4141 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4201 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4261 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4321 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4381 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4441 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4501 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4561 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4621 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4681 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4741 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4801 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4861 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4921 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4981 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5041 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5101 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5161 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5221 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5281 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5341 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5401 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5461 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5521 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5581 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5641 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5701 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5761 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     5821 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     5881 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     5941 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     6001 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     6061 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     6121 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     6181 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     6241 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
     6301 gggaagggcg atcggtgcgg gcctcttcgc tattacgc
//
