LOCUS       sequence-183021-        6380 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of Chronos-GFP under the Syn promoter, in 
            floxed/forward (Cre-dependent) manner. Cre turns gene off. Using 
            bGHpA signal..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6380)
  AUTHORS   Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK,
            Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, 
            Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, 
            Constantine-Paton M, Wong GK, Boyden ES
  TITLE     Independent optical excitation of distinct neural populations.
  JOURNAL   Nat Methods. 2014 Mar;11(3):338-46. doi: 10.1038/nmeth.2836. Epub 
            2014 Feb 9.
  PUBMED    24509633
REFERENCE   2  (bases 1 to 6380)
  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"; doi: 
            "10.1038/nmeth.2836"; journalName: "Nat Methods"; date: "2014-03"; 
            volume: "11"; issue: "3"; pages: "338-46"
COMMENT     Sequence Label: pAAV-Syn-FLEX-fd [Chronos-GFP]
FEATURES             Location/Qualifiers
     source          1..6380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      296..305
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(316..349)
                     /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(412..445)
                     /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(461..908)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   1011..1140
                     /label=AAV2 ITR
     rep_origin      1212..1667
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1299..1318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1509..1530
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1693..1797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1798..1866,1867..2658)
                     /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(2016..2035)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2829..3417
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3318..3337
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3654..3783
                     /label=AAV2 ITR
     polyA_signal    3953..4160
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    4201..4789
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             4307..4318
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4716..4736
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    4822..4855
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4926..4959
                     /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(4970..5683,5684..5686))
                     /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(4973..5683,5684..5686))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5015..5036)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5364..5383
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5623..5644
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 tcctcccagc cggttgtggc cttgaagctg tgccaaccgt agaagatgag ctgcacgata
       61 gccacgatca cggtgaacca ctggagtccc actgcgatct tgtgctgcag ctcggttccg
      121 tggttgatgt gggggaagca accgtgatcg gctccggctg ttgtggcgtt agaggtttct
      181 ccgtgagcgt ctgctgctgg tgtcaccact gctgcggcgc tcagcaggcc tctaattgtg
      241 gcttcggcgc taggcacggc tgagataaag gcgtgggtca ttgtggcggc tgtttccatg
      301 gtggcgctag ctagtataac ttcgtatagc atacattata cgaagttatg ggtcgatggt
      361 gatgcttggc aattcgggtc gatggtgaag cattggtctt cctattttga tataacttcg
      421 tataggatac tttatacgaa gttatgggaa ttcttctcga ctgcgctctc aggcacgaca
      481 cgactcctcc gctgcccacc gcagactgag gcagcgctga gtcgccggcg ccgcagcgca
      541 gatggtcgcg cccgtgcccc cctatctcgc gcctcgcgtg gtgcggtccg gctgggccgg
      601 cggcggcgcg gacgcgacca aggtggccgg gaaggggagt ttgcggggga ccggcgagtg
      661 acgtcagcgc gccttcagtg ctgaggcggc ggtggcgcgc gccgccaggc gggggcgaag
      721 gcactgtccg cggtgctgaa gctggcagtg cgcacgcgcc tcgccgcatc ctgtttcccc
      781 tccccctctc tgatagggga tgcgcaattt ggggaatggg ggttgggtgc ttgtccagtg
      841 ggtcggggtc ggtcgtcagg taggcacccc caccccgcct catcctggtc ctaaaaccca
      901 cttgcactca tacgcagggc cctctgcagt ctagacactt aattaaccta cgatcttcct
      961 agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct
     1021 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     1081 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     1141 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     1201 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1261 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1321 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1381 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1441 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1501 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1561 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1621 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     1681 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1741 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1801 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1861 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1921 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1981 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2041 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     2101 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     2161 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     2221 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     2281 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2341 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2401 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2461 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2521 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2581 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2641 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     2701 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2761 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2821 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2881 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2941 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     3001 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3061 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3121 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3181 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3241 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3301 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3361 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3421 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3481 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3541 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3601 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgcgcg
     3661 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
     3721 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     3781 ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga
     3841 tccttatcga ttttaccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacatc
     3901 tccccctgaa cctgaaacat aaaatgaatg caattgtaca gtccggaccg catccccagc
     3961 atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
     4021 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
     4081 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
     4141 ctggcaacta gaaggcacag tcgagcttta tgagcggccg cccgggtcga cggtatcgat
     4201 gcggggaggc ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa
     4261 gaggccgcag agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag
     4321 ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccaag
     4381 gaaaggacga tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc
     4441 ctgtccagca gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga
     4501 aagcgaaagt cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg
     4561 cgtcagcaaa cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat
     4621 aaagagacag caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa
     4681 tagcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt
     4741 taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgatta tcgataagct
     4801 tcgcgaacct cttcgaggga cataacttcg tataatgtat gctatacgaa gttatgggtc
     4861 gatggtgaga tctggactag agggtcgatg gtgatgcttg gatcaaaata ggaagaatcc
     4921 caatgataac ttcgtataaa gtatcctata cgaagttata ctaggtacct tacttgtaca
     4981 gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat
     5041 cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg
     5101 gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca
     5161 cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct
     5221 tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga
     5281 tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc
     5341 cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc
     5401 gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt
     5461 cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg
     5521 gcacgggcag cttgccggtg gtgcaaatga acttcagggt cagcttgccg taggtggcat
     5581 cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga
     5641 ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcactgct actaccggtg
     5701 ccgccactcc tccctcctcc tcctcgtcga cgaaggtctc gatctccatg ttctcgccgg
     5761 ccacgttgat ggtggttgtc ttccggatgt cgccgtggat caggatgtgc tcgtggatct
     5821 tcactctcag gtggtggccc aggaagcccc aagtgttctt gctgatcaga tccaggatca
     5881 ggtggccgat tccgctggtg taaggtgtga tcaggcccag tccctcgtgt ccggcgatga
     5941 acatcacggg gaacatcagc caagagcaga agaagacgta ggccatgatc ttgcaaatct
     6001 tccggcagac gcctttgggc agggtgtggt agctctcgat atacaccttg gcgatctgga
     6061 agaagcaagt cacgccgtag aacaggccga tcatgaagaa caggatcttc agggggccct
     6121 ttgtaaaggc ggctgtgatc ccccacacga tgttgccgat gtcggtcacc aggatggtca
     6181 tggtccgctt gctgtactct tcgtgcagtc cggtcaggtt gctcaggtgg atcaggatca
     6241 cggggcaagt caggagccac atgctgtacc gcagccaaat cacggctcct ccgttggtct
     6301 ggtacactgt ggctgggctg tcgacctcgt ggaacagctc gatgaagcac ttgacgagct
     6361 cgatcacgca gacgtagacc
//
