LOCUS       sequence-257673-        6866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of Chronos-GFP under the EF1-alpha-1.1 
            promoter in frt/reversed (Flp-dependent) manner..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6866)
  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 6866)
  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-FLPX-rc [Chronos-GFP]
FEATURES             Location/Qualifiers
     source          1..6866
                     /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        145..1323
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          376..1314
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1271..1291
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1330..1363
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(join(1457..2170,2171..2173))
                     /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(1460..2170,2171..2173))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1502..1523)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1851..1870
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2110..2131
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3163..3172
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3190..3223)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    3313..3901
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3366..3386)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3784..3795)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   4129..4269
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4129..4258
                     /label=AAV2 ITR
     rep_origin      4344..4799
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4431..4450)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4641..4662
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4816..4835)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4935..4957
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4995..5013)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5081..5185
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5186..5254,5255..6046)
                     /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(5404..5423)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6217..6805
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6706..6725
                     /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 gtcgacttaa ttaaggctcc ggtgcccgtc agtgggcaga gcgcacatcg
      181 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
      241 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
      301 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
      361 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
      421 tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc
      481 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
      541 cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg
      601 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
      661 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
      721 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
      781 tgttcgacga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
      841 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
      901 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
      961 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
     1021 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     1081 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     1141 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     1201 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     1261 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     1321 tgagctagcg aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga
     1381 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     1441 gaacttcgaa ttctcattac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
     1501 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg
     1561 actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
     1621 tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga
     1681 agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt
     1741 agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
     1801 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
     1861 tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact
     1921 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
     1981 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg caaatgaact
     2041 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
     2101 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
     2161 cgcccttgct cactgctact accggtgccg ccactcctcc ctcctcctcc tcgtcgacga
     2221 aggtctcgat ctccatgttc tcgccggcca cgttgatggt ggttgtcttc cggatgtcgc
     2281 cgtggatcag gatgtgctcg tggatcttca ctctcaggtg gtggcccagg aagccccaag
     2341 tgttcttgct gatcagatcc aggatcaggt ggccgattcc gctggtgtaa ggtgtgatca
     2401 ggcccagtcc ctcgtgtccg gcgatgaaca tcacggggaa catcagccaa gagcagaaga
     2461 agacgtaggc catgatcttg caaatcttcc ggcagacgcc tttgggcagg gtgtggtagc
     2521 tctcgatata caccttggcg atctggaaga agcaagtcac gccgtagaac aggccgatca
     2581 tgaagaacag gatcttcagg gggccctttg taaaggcggc tgtgatcccc cacacgatgt
     2641 tgccgatgtc ggtcaccagg atggtcatgg tccgcttgct gtactcttcg tgcagtccgg
     2701 tcaggttgct caggtggatc aggatcacgg ggcaagtcag gagccacatg ctgtaccgca
     2761 gccaaatcac ggctcctccg ttggtctggt acactgtggc tgggctgtcg acctcgtgga
     2821 acagctcgat gaagcacttg acgagctcga tcacgcagac gtagacctcc tcccagccgg
     2881 ttgtggcctt gaagctgtgc caaccgtaga agatgagctg cacgatagcc acgatcacgg
     2941 tgaaccactg gagtcccact gcgatcttgt gctgcagctc ggttccgtgg ttgatgtggg
     3001 ggaagcaacc gtgatcggct ccggctgttg tggcgttaga ggtttctccg tgagcgtctg
     3061 ctgctggtgt caccactgct gcggcgctca gcaggcctct aattgtggct tcggcgctag
     3121 gcacggctga gataaaggcg tgggtcattg tggcggctgt ttccatggtg gcggatccgg
     3181 cggcgcgccg aagttcctat actttctaga gaataggaac ttcttgcctt aacccagaaa
     3241 ttatcactgt tattctttag aatggtgcaa agagaagttc ctataccttt tgaagaatag
     3301 gaacttctcc ggaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3361 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3421 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3481 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3541 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3601 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     3661 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt
     3721 ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     3781 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     3841 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     3901 ccttgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3961 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4021 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4081 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggcag gaacccctag
     4141 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4201 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4261 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4321 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4381 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4441 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4501 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4561 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4621 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     4681 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     4741 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     4801 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4861 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4921 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4981 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5041 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5101 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5161 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5221 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5281 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5341 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5401 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5461 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5521 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5581 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5641 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5701 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5761 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5821 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5881 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5941 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6001 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6061 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6121 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6181 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6241 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6301 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6361 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6421 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6481 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6541 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6601 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6661 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6721 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6781 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6841 cttttgctgg ccttttgctc acatgt
//
