LOCUS       sequence-103556-        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"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     promoter        2960..4150
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3195..4141
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4098..4118
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    4170..4203
                     /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    4266..4299
                     /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(4313..5026,5027..5029))
                     /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(4316..5026,5027..5029))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4358..4379)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4707..4726
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4966..4987
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      6019..6028
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(6050..6083)
                     /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(6154..6187)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6220..6808
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6273..6293)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6691..6702)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6849..7056
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7226..7355
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagatcgta ggttaattaa gcagatctcg accaattctc atgtttgaca gcttatcatc
     2701 gatcagcttt ggagctaagc cagcaatggt agagggaaga ttctgcacgt cccttccagg
     2761 cggcctcccc gtcaccaccc cccccaaccc gccccgaccg gagctgagag taattcatac
     2821 aaaaggactc gcccctgcct tggggaatcc cagggaccgt cgttaaactc ccactaacgt
     2881 agaacccaga gatcgctgcg ttcccgcccc ctcacccgcc cgctctcgtc atcactgagg
     2941 tggagaagag catgcgtgag gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     3001 agtccccgag aagttggggg gaggggtcgg caattgaacc ggccggtgcc tagagaaagt
     3061 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     3121 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     3181 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
     3241 atggcccttg cgtgccttga attacttcca cgcccctggc tgcagtacgt gattcttgat
     3301 cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct
     3361 tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg
     3421 tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga
     3481 tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg
     3541 cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc
     3601 acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct
     3661 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg
     3721 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc
     3781 cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca
     3841 cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag
     3901 taccgggcgc cgtccaggca cctcgattag ttctcgatcg agcttttgga gtacgtcgtc
     3961 tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac
     4021 tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt
     4081 tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca
     4141 ggtgtcgtga aaactctagg agaattccca taacttcgta taaagtatcc tatacgaagt
     4201 tatatcaaaa taggaagacc aatgcttcac catcgacccg aattgccaag catcaccatc
     4261 gacccataac ttcgtataat gtatgctata cgaagttata ctagctagct cattacttgt
     4321 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
     4381 gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt
     4441 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct
     4501 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct
     4561 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca
     4621 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     4681 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     4741 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     4801 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
     4861 agggcacggg cagcttgccg gtggtgcaaa tgaacttcag ggtcagcttg ccgtaggtgg
     4921 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
     4981 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcact gctactaccg
     5041 gtgccgccac tcctccctcc tcctcctcgt cgacgaaggt ctcgatctcc atgttctcgc
     5101 cggccacgtt gatggtggtt gtcttccgga tgtcgccgtg gatcaggatg tgctcgtgga
     5161 tcttcactct caggtggtgg cccaggaagc cccaagtgtt cttgctgatc agatccagga
     5221 tcaggtggcc gattccgctg gtgtaaggtg tgatcaggcc cagtccctcg tgtccggcga
     5281 tgaacatcac ggggaacatc agccaagagc agaagaagac gtaggccatg atcttgcaaa
     5341 tcttccggca gacgcctttg ggcagggtgt ggtagctctc gatatacacc ttggcgatct
     5401 ggaagaagca agtcacgccg tagaacaggc cgatcatgaa gaacaggatc ttcagggggc
     5461 cctttgtaaa ggcggctgtg atcccccaca cgatgttgcc gatgtcggtc accaggatgg
     5521 tcatggtccg cttgctgtac tcttcgtgca gtccggtcag gttgctcagg tggatcagga
     5581 tcacggggca agtcaggagc cacatgctgt accgcagcca aatcacggct cctccgttgg
     5641 tctggtacac tgtggctggg ctgtcgacct cgtggaacag ctcgatgaag cacttgacga
     5701 gctcgatcac gcagacgtag acctcctccc agccggttgt ggccttgaag ctgtgccaac
     5761 cgtagaagat gagctgcacg atagccacga tcacggtgaa ccactggagt cccactgcga
     5821 tcttgtgctg cagctcggtt ccgtggttga tgtgggggaa gcaaccgtga tcggctccgg
     5881 ctgttgtggc gttagaggtt tctccgtgag cgtctgctgc tggtgtcacc actgctgcgg
     5941 cgctcagcag gcctctaatt gtggcttcgg cgctaggcac ggctgagata aaggcgtggg
     6001 tcattgtggc ggctgtttcc atggtggcgg cgcggcctag gtacctagta taacttcgta
     6061 taggatactt tatacgaagt tatcattggg attcttccta ttttgatcca agcatcacca
     6121 tcgaccctct agtccagatc tcaccatcga cccataactt cgtatagcat acattatacg
     6181 aagttatgtc cctcgaagag gttcgcgaag cttatcgata atcaacctct ggattacaaa
     6241 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     6301 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     6361 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     6421 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     6481 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     6541 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     6601 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     6661 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     6721 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     6781 cggatctccc tttgggccgc ctccccgcat cgataccgtc gacccgggcg gccgctcata
     6841 aagctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     6901 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     6961 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     7021 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtccggactg tacaattgca
     7081 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc
     7141 tctacaaatg tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag
     7201 catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct
     7261 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     7321 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg
     7381 cggggagagg cggtttgcgt attgggcgct
//
