LOCUS       sequence-242267-        6684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of Chronos-GFP under the EF1-alpha promoter 
            (1.1kb short version). Using SV40 pA signal..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6684)
  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 6684)
  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-Chronos-GFP
FEATURES             Location/Qualifiers
     source          1..6684
                     /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        2661..3839
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2892..3830
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3787..3807
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3846..3855
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4845..4847,4848..5561)
                     /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(4845..4847,4848..5558)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4887..4908)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5148..5167)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5495..5516
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5586..6174
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5639..5659)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6057..6068)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6217..6338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6228..6247)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6282..6301
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6500..6629
                     /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 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     2701 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
     2761 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     2821 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     2881 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
     2941 gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc
     3001 ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc
     3061 gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc
     3121 ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg
     3181 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg
     3241 gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt
     3301 cgacgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg
     3361 gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa
     3421 ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg
     3481 cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac
     3541 aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg
     3601 cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg
     3661 gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc
     3721 cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt
     3781 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag
     3841 gatccgccac catggaaaca gccgccacaa tgacccacgc ctttatctca gccgtgccta
     3901 gcgccgaagc cacaattaga ggcctgctga gcgccgcagc agtggtgaca ccagcagcag
     3961 acgctcacgg agaaacctct aacgccacaa cagccggagc cgatcacggt tgcttccccc
     4021 acatcaacca cggaaccgag ctgcagcaca agatcgcagt gggactccag tggttcaccg
     4081 tgatcgtggc tatcgtgcag ctcatcttct acggttggca cagcttcaag gccacaaccg
     4141 gctgggagga ggtctacgtc tgcgtgatcg agctcgtcaa gtgcttcatc gagctgttcc
     4201 acgaggtcga cagcccagcc acagtgtacc agaccaacgg aggagccgtg atttggctgc
     4261 ggtacagcat gtggctcctg acttgccccg tgatcctgat ccacctgagc aacctgaccg
     4321 gactgcacga agagtacagc aagcggacca tgaccatcct ggtgaccgac atcggcaaca
     4381 tcgtgtgggg gatcacagcc gcctttacaa agggccccct gaagatcctg ttcttcatga
     4441 tcggcctgtt ctacggcgtg acttgcttct tccagatcgc caaggtgtat atcgagagct
     4501 accacaccct gcccaaaggc gtctgccgga agatttgcaa gatcatggcc tacgtcttct
     4561 tctgctcttg gctgatgttc cccgtgatgt tcatcgccgg acacgaggga ctgggcctga
     4621 tcacacctta caccagcgga atcggccacc tgatcctgga tctgatcagc aagaacactt
     4681 ggggcttcct gggccaccac ctgagagtga agatccacga gcacatcctg atccacggcg
     4741 acatccggaa gacaaccacc atcaacgtgg ccggcgagaa catggagatc gagaccttcg
     4801 tcgacgagga ggaggaggga ggagtggcgg caccggtagt agcagtgagc aagggcgagg
     4861 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     4921 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     4981 tcatttgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     5041 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     5101 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     5161 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     5221 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     5281 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     5341 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     5401 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     5461 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     5521 ccgccgggat cactctcggc atggacgagc tgtacaagta agaattcgat atcaagctta
     5581 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     5641 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     5701 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     5761 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     5821 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     5881 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     5941 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     6001 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     6061 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     6121 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     6181 accgtcgacc cgggcggccg cttcgagcag acatgataag atacattgat gagtttggac
     6241 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     6301 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     6361 ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa aacctctaca
     6421 aatgtggtaa aatcgataag gatcttccta gagcatggct acgtagataa gtagcatggc
     6481 gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     6541 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     6601 gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg aatcggccaa cgcgcgggga
     6661 gaggcggttt gcgtattggg cgct
//
