LOCUS       sequence-193534-        6371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of Chronos-GFP under the CAG promoter. Using
            bGHpA signal. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6371)
  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 6371)
  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-CAG-Chronos-GFP
FEATURES             Location/Qualifiers
     source          1..6371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        387..664
                     /label=chicken beta-actin promoter
     primer_bind     943..965
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1027..1046
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1082..1091
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2081..2083,2084..2797)
                     /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(2081..2083,2084..2794)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2123..2144)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2384..2403)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2731..2752
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2812..3400
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2865..2885)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3283..3294)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3408..3632
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3634..3774
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3634..3763
                     /label=AAV2 ITR
     rep_origin      3849..4304
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3936..3955)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4146..4167
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4321..4340)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4440..4462
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4500..4518)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4586..4690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4691..4759,4760..5551)
                     /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(4909..4928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5722..6310
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6211..6230
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gtcgacccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      181 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      241 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      301 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      361 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
      421 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
      481 agcgatgggg gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg
      541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggctgtc
      901 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
      961 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1021 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgaattcgc
     1081 cgccaccatg gaaacagccg ccacaatgac ccacgccttt atctcagccg tgcctagcgc
     1141 cgaagccaca attagaggcc tgctgagcgc cgcagcagtg gtgacaccag cagcagacgc
     1201 tcacggagaa acctctaacg ccacaacagc cggagccgat cacggttgct tcccccacat
     1261 caaccacgga accgagctgc agcacaagat cgcagtggga ctccagtggt tcaccgtgat
     1321 cgtggctatc gtgcagctca tcttctacgg ttggcacagc ttcaaggcca caaccggctg
     1381 ggaggaggtc tacgtctgcg tgatcgagct cgtcaagtgc ttcatcgagc tgttccacga
     1441 ggtcgacagc ccagccacag tgtaccagac caacggagga gccgtgattt ggctgcggta
     1501 cagcatgtgg ctcctgactt gccccgtgat cctgatccac ctgagcaacc tgaccggact
     1561 gcacgaagag tacagcaagc ggaccatgac catcctggtg accgacatcg gcaacatcgt
     1621 gtgggggatc acagccgcct ttacaaaggg ccccctgaag atcctgttct tcatgatcgg
     1681 cctgttctac ggcgtgactt gcttcttcca gatcgccaag gtgtatatcg agagctacca
     1741 caccctgccc aaaggcgtct gccggaagat ttgcaagatc atggcctacg tcttcttctg
     1801 ctcttggctg atgttccccg tgatgttcat cgccggacac gagggactgg gcctgatcac
     1861 accttacacc agcggaatcg gccacctgat cctggatctg atcagcaaga acacttgggg
     1921 cttcctgggc caccacctga gagtgaagat ccacgagcac atcctgatcc acggcgacat
     1981 ccggaagaca accaccatca acgtggccgg cgagaacatg gagatcgaga ccttcgtcga
     2041 cgaggaggag gagggaggag tggcggcacc ggtagtagca gtgagcaagg gcgaggagct
     2101 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     2161 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     2221 ttgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     2281 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2341 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2401 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2461 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2521 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     2581 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2641 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     2701 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2761 cgggatcact ctcggcatgg acgagctgta caagtaatga gatatctccg gaatcaacct
     2821 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     2881 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     2941 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     3001 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     3061 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     3121 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     3181 gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt
     3241 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     3301 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     3361 cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggatcctg tgccttctag
     3421 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3481 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3541 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3601 caggcatgct ggggatgcgg tgggctctat ggcaggaacc cctagtgatg gagttggcca
     3661 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3721 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     3781 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     3841 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3901 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     3961 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4021 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     4081 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4141 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     4201 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     4261 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     4321 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4381 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4441 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4501 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4561 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4621 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4681 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4741 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4801 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4861 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4921 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4981 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5041 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5101 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5161 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5221 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5281 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5341 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5401 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5461 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5521 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5581 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5641 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5701 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5761 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5821 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5881 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5941 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6001 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6061 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6121 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6181 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6241 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6301 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6361 tgctcacatg t
//
