LOCUS       sequence-169762-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of Chronos-GFP under the EF1-alpha promoter,
            in floxed/reversed (Cre-dependent) manner. Using bGHpA signal..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  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 7410)
  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-EF1-alpha-FLEX-rc[Chronos-GFP]
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   55..184
                     /label=AAV2 ITR
     rep_origin      complement(421..1009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(501..520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1180..1971,1972..2040))
                     /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     1803..1822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2041..2145)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2171..2626)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2308..2329)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2520..2539
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2698..2827
                     /label=AAV2 ITR
     promoter        3199..4389
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3434..4380
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4337..4357
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    4409..4442
                     /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    4505..4538
                     /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(4552..5265,5266..5268))
                     /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(4555..5265,5266..5268))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4597..4618)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4946..4965
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5205..5226
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      6258..6267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(6289..6322)
                     /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(6393..6426)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6459..7047
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6512..6532)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6930..6941)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7088..7295
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac
       61 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
      121 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
      181 gcagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
      241 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
      301 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
      361 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      421 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      481 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      541 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      601 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      661 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      721 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      781 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      841 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      901 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      961 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1021 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1081 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1141 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1201 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1261 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1321 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1381 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1441 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1501 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1561 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1621 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1681 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1741 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1801 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1861 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1921 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1981 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2041 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2101 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2161 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     2221 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     2281 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     2341 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     2401 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     2461 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     2521 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     2581 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
     2641 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     2701 cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt
     2761 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
     2821 ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc atgctctagg
     2881 aagatcgtag gttaattaag cagatctcga ccaattctca tgtttgacag cttatcatcg
     2941 atcagctttg gagctaagcc agcaatggta gagggaagat tctgcacgtc ccttccaggc
     3001 ggcctccccg tcaccacccc ccccaacccg ccccgaccgg agctgagagt aattcataca
     3061 aaaggactcg cccctgcctt ggggaatccc agggaccgtc gttaaactcc cactaacgta
     3121 gaacccagag atcgctgcgt tcccgccccc tcacccgccc gctctcgtca tcactgaggt
     3181 ggagaagagc atgcgtgagg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     3241 gtccccgaga agttgggggg aggggtcggc aattgaaccg gccggtgcct agagaaagtg
     3301 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     3361 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     3421 cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta
     3481 tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg attcttgatc
     3541 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
     3601 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
     3661 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
     3721 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc
     3781 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
     3841 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
     3901 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
     3961 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
     4021 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac
     4081 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     4141 accgggcgcc gtccaggcac ctcgattagt tctcgatcga gcttttggag tacgtcgtct
     4201 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact
     4261 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt
     4321 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag
     4381 gtgtcgtgaa aactctagga gaattcccat aacttcgtat aaagtatcct atacgaagtt
     4441 atatcaaaat aggaagacca atgcttcacc atcgacccga attgccaagc atcaccatcg
     4501 acccataact tcgtataatg tatgctatac gaagttatac tagctagctc attacttgta
     4561 cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg
     4621 atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc
     4681 gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg
     4741 cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg
     4801 cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag
     4861 gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc
     4921 gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt
     4981 gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg
     5041 gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca
     5101 gggcacgggc agcttgccgg tggtgcaaat gaacttcagg gtcagcttgc cgtaggtggc
     5161 atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc
     5221 gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcactg ctactaccgg
     5281 tgccgccact cctccctcct cctcctcgtc gacgaaggtc tcgatctcca tgttctcgcc
     5341 ggccacgttg atggtggttg tcttccggat gtcgccgtgg atcaggatgt gctcgtggat
     5401 cttcactctc aggtggtggc ccaggaagcc ccaagtgttc ttgctgatca gatccaggat
     5461 caggtggccg attccgctgg tgtaaggtgt gatcaggccc agtccctcgt gtccggcgat
     5521 gaacatcacg gggaacatca gccaagagca gaagaagacg taggccatga tcttgcaaat
     5581 cttccggcag acgcctttgg gcagggtgtg gtagctctcg atatacacct tggcgatctg
     5641 gaagaagcaa gtcacgccgt agaacaggcc gatcatgaag aacaggatct tcagggggcc
     5701 ctttgtaaag gcggctgtga tcccccacac gatgttgccg atgtcggtca ccaggatggt
     5761 catggtccgc ttgctgtact cttcgtgcag tccggtcagg ttgctcaggt ggatcaggat
     5821 cacggggcaa gtcaggagcc acatgctgta ccgcagccaa atcacggctc ctccgttggt
     5881 ctggtacact gtggctgggc tgtcgacctc gtggaacagc tcgatgaagc acttgacgag
     5941 ctcgatcacg cagacgtaga cctcctccca gccggttgtg gccttgaagc tgtgccaacc
     6001 gtagaagatg agctgcacga tagccacgat cacggtgaac cactggagtc ccactgcgat
     6061 cttgtgctgc agctcggttc cgtggttgat gtgggggaag caaccgtgat cggctccggc
     6121 tgttgtggcg ttagaggttt ctccgtgagc gtctgctgct ggtgtcacca ctgctgcggc
     6181 gctcagcagg cctctaattg tggcttcggc gctaggcacg gctgagataa aggcgtgggt
     6241 cattgtggcg gctgtttcca tggtggcggc gcggcctagg tacctagtat aacttcgtat
     6301 aggatacttt atacgaagtt atcattggga ttcttcctat tttgatccaa gcatcaccat
     6361 cgaccctcta gtccagatct caccatcgac ccataacttc gtatagcata cattatacga
     6421 agttatgtcc ctcgaagagg ttcgcgaagc ttatcgataa tcaacctctg gattacaaaa
     6481 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     6541 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     6601 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     6661 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     6721 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     6781 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     6841 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     6901 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     6961 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     7021 ggatctccct ttgggccgcc tccccgcatc gataccgtcg acccgggcgg ccgctcataa
     7081 agctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     7141 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     7201 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     7261 gaggattggg aagacaatag caggcatgct ggggatgcgg tccggactgt acaattgcat
     7321 tcattttatg tttcaggttc agggggagat gtgggaggtt ttttaaagca agtaaaacct
     7381 ctacaaatgt ggtaaaatcg ataaggatct
//
