LOCUS       sequence-176951-        7088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of Chronos-GFP under the EF1-alpha promoter 
            (1.1kb short version), 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 7088)
  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 7088)
  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-1.1-FLEX-rc [Chronos-GFP]
FEATURES             Location/Qualifiers
     source          1..7088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   3..132
                     /label=AAV2 ITR
     polyA_signal    302..509
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    550..1138
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             656..667
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1065..1085
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1171..1204
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1275..1308
                     /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)."
     regulatory      1330..1339
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2329..2331,2332..3045)
                     /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             join(2329..2331,2332..3042)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2371..2392)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2632..2651)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2979..3000
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3059..3092)
                     /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(3155..3188)
                     /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(3197..4375)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3206..4144
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3229..3249)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   4448..4577
                     /label=AAV2 ITR
     rep_origin      4649..5104
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4736..4755)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4946..4967
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5130..5234
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5235..5303,5304..6095)
                     /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(5453..5472)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6266..6854
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6755..6774
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct
       61 ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca
      121 ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctacg tagccatgct
      181 ctaggaagat ccttatcgat tttaccacat ttgtagaggt tttacttgct ttaaaaaacc
      241 tcccacatct ccccctgaac ctgaaacata aaatgaatgc aattgtacag tccggaccgc
      301 atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct gccccacccc
      361 accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct cattttatta
      421 ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg aggggcaaac
      481 aacagatggc tggcaactag aaggcacagt cgagctttat gagcggccgc ccgggtcgac
      541 ggtatcgatg cggggaggcg gcccaaaggg agatccgact cgtctgaggg cgaaggcgaa
      601 gacgcggaag aggccgcaga gccggcagca ggccgcggga aggaaggtcc gctggattga
      661 gggccgaagg gacgtagcag aaggacgtcc cgcgcagaat ccaggtggca acacaggcga
      721 gcagccaagg aaaggacgat gatttccccg acaacaccac ggaattgtca gtgcccaaca
      781 gccgagcccc tgtccagcag cgggcaaggc aggcggcgat gagttccgcc gtggcaatag
      841 ggagggggaa agcgaaagtc ccggaaagga gctgacaggt ggtggcaatg ccccaaccag
      901 tgggggttgc gtcagcaaac acagtgcaca ccacgccacg ttgcctgaca acgggccaca
      961 actcctcata aagagacagc aaccaggatt tatacaagga ggagaaaatg aaagccatac
     1021 gggaagcaat agcatgatac aaaggcatta aagcagcgta tccacatagc gtaaaaggag
     1081 caacatagtt aagaatacca gtcaatcttt cacaaatttt gtaatccaga ggttgattat
     1141 cgataagctt cgcgaacctc ttcgagggac ataacttcgt ataatgtatg ctatacgaag
     1201 ttatgggtcg atggtgagat ctggactaga gggtcgatgg tgatgcttgg atcaaaatag
     1261 gaagaatccc aatgataact tcgtataaag tatcctatac gaagttatac taggtaccta
     1321 ggccgcgccg ccaccatgga aacagccgcc acaatgaccc acgcctttat ctcagccgtg
     1381 cctagcgccg aagccacaat tagaggcctg ctgagcgccg cagcagtggt gacaccagca
     1441 gcagacgctc acggagaaac ctctaacgcc acaacagccg gagccgatca cggttgcttc
     1501 ccccacatca accacggaac cgagctgcag cacaagatcg cagtgggact ccagtggttc
     1561 accgtgatcg tggctatcgt gcagctcatc ttctacggtt ggcacagctt caaggccaca
     1621 accggctggg aggaggtcta cgtctgcgtg atcgagctcg tcaagtgctt catcgagctg
     1681 ttccacgagg tcgacagccc agccacagtg taccagacca acggaggagc cgtgatttgg
     1741 ctgcggtaca gcatgtggct cctgacttgc cccgtgatcc tgatccacct gagcaacctg
     1801 accggactgc acgaagagta cagcaagcgg accatgacca tcctggtgac cgacatcggc
     1861 aacatcgtgt gggggatcac agccgccttt acaaagggcc ccctgaagat cctgttcttc
     1921 atgatcggcc tgttctacgg cgtgacttgc ttcttccaga tcgccaaggt gtatatcgag
     1981 agctaccaca ccctgcccaa aggcgtctgc cggaagattt gcaagatcat ggcctacgtc
     2041 ttcttctgct cttggctgat gttccccgtg atgttcatcg ccggacacga gggactgggc
     2101 ctgatcacac cttacaccag cggaatcggc cacctgatcc tggatctgat cagcaagaac
     2161 acttggggct tcctgggcca ccacctgaga gtgaagatcc acgagcacat cctgatccac
     2221 ggcgacatcc ggaagacaac caccatcaac gtggccggcg agaacatgga gatcgagacc
     2281 ttcgtcgacg aggaggagga gggaggagtg gcggcaccgg tagtagcagt gagcaagggc
     2341 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2401 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2461 aagttcattt gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     2521 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     2581 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2641 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2701 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2761 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     2821 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     2881 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     2941 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3001 accgccgccg ggatcactct cggcatggac gagctgtaca agtaatgagc tagctagtat
     3061 aacttcgtat agcatacatt atacgaagtt atgggtcgat ggtgatgctt ggcaattcgg
     3121 gtcgatggtg aagcattggt cttcctattt tgatataact tcgtatagga tactttatac
     3181 gaagttatgg gaattctcac gacacctgaa atggaagaaa aaaactttga accactgtct
     3241 gaggcttgag aatgaaccaa gatccaaact caaaaagggc aaattccaag gagaattaca
     3301 tcaagtgcca agctggccta acttcagtct ccacccactc agtgtgggga aactccatcg
     3361 cataaaaccc ctccccccaa cctaaagacg acgtactcca aaagctcgag aactaatcga
     3421 ggtgcctgga cggcgcccgg tactccgtgg agtcacatga agcgacggct gaggacggaa
     3481 aggccctttt cctttgtgtg ggtgactcac ccgcccgctc tcccgagcgc cgcgtcctcc
     3541 attttgagct ccctgcagca gggccgggaa gcggccatct ttccgctcac gcaactggtg
     3601 ccgaccgggc cagccttgcc gcccagggcg gggcgataca cggcggcgcg aggccaggca
     3661 ccagagcagg ccggccagct tgagactacc cccgtccgat tctcggtggc cgcgctcgca
     3721 ggccccgcct cgccgaacat gtgcgctggg acgcacgggc cccgtcgccg cccgcggccc
     3781 caaaaaccga aataccagtg tgcagatctt ggcccgcatt tacaagacta tcttgccaga
     3841 aaaaaagcgt cgcagcaggt catcaaaaat tttaaatggc tagagactta tcgaaagcag
     3901 cgagacaggc gcgaaggtgc caccagattc gcacgcggcg gccccagcgc ccaggccagg
     3961 cctcaactca agcacgaggc gaaggggctc cttaagcgca aggcctcgaa ctctcccacc
     4021 cacttccaac ccgaagctcg ggatcaagaa tcacgtactg cagccaggtg gaagtaattc
     4081 aaggcacgca agggccataa cccgtaaaga ggccaggccc gcgggaacca cacacggcac
     4141 ttacctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac
     4201 ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt
     4261 cccagtttac cccgcgccac cttctctagg caccggttca attgccgacc cctcccccca
     4321 acttctcggg gactgtgggc gatgtgcgct ctgcccactg acgggcaccg gagccttaat
     4381 taacctacga tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt
     4441 aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4501 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4561 agcgagcgag cgcgcagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4621 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     4681 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4741 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4801 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4861 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4921 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4981 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5041 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5101 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5161 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5221 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5281 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5341 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5401 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5461 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5521 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5581 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5641 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5701 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5761 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5821 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5881 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5941 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6001 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6061 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6121 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6181 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6241 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6301 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6361 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6421 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6481 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6541 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6601 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6661 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6721 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6781 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6841 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6901 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6961 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     7021 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     7081 attaatgc
//
