LOCUS       sequence-256678-        7577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of Chronos-tdTomato under the EF1-alpha-1.1 
            promoter in frt/reversed (Flp-dependent) manner. tdTomato has codons
            varied to reduce recombination..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7577)
  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 7577)
  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: AAV-EF1-alpha-1.1-FLPX-rc [Chronos-tdTomato]
FEATURES             Location/Qualifiers
     source          1..7577
                     /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)"
     regulatory      3874..3883
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3901..3934)
                     /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    4024..4612
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4077..4097)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4495..4506)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   4840..4980
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4840..4969
                     /label=AAV2 ITR
     rep_origin      5055..5510
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5142..5161)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5352..5373
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5527..5546)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5646..5668
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5706..5724)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5792..5896
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5897..5965,5966..6757)
                     /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(6115..6134)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6928..7516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7417..7436
                     /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 ttatacagct catccatgcc gtacagaaac agatggtggc
     1501 gtccttcaga cctctcatac tgttccacaa tggtgtagtc ttcattgtgg gatgtgatat
     1561 ccagcttggt gtccacatag taatagccgg gcagctgcac tggtttcttg gccatgtaga
     1621 tggttttgaa ctccaccagg tagtggccgc cgtctttcag cttcagggcc tggtggattt
     1681 cgcccttcag cactccgtct ctaggataca gccgttcagt gctggcctcc caccccattg
     1741 ttttcttctg catcacgggc ccatcgggtg gaaaattggt gccgcgcatt ttgactttgt
     1801 agatcagagt gccatcctgc agagaggagt cctgtgtcac ggtcaccagg cctccatctt
     1861 caaaattcat cactctctcc cacttaaagc cctctgggaa agacagtttc ttgtaatcag
     1921 gaatgtcagc gggatgcttg acataggcct tacttccgta cataaactga ggagacagga
     1981 tatcccaagc gaaaggcagt ggtcctccct tggtcacttt cagcttggca gtctgtgtgc
     2041 cctcgtaagg cctcccttcg ccctctcctt caatctcaaa ttcgtgccca ttcatgctgc
     2101 cctccatgcg caccttaaac ctcatgaact ctttgatcac agccatattg ttatcctcgc
     2161 ttgaggcggt tccactagac cctgagccgg tacttccagt cccatggccc aggaacagat
     2221 ggtgtctccc ctcgctccgt tcatactgct ccacaatagt gtagtcctcg ttgtgtgaag
     2281 tgatatccag ttttgtgtcc acgtaatagt agcctggcag ctgcacaggt ttcttggcca
     2341 tgtagattgt cttgaactcc accaggtaat gtcccccatc tttcagcttc agagcctggt
     2401 ggatttctcc tttcagcacc ccgtcgcgtg gatacagcct ttcggtggag gcctcccatc
     2461 ccatagtttt cttctgcatc acaggtccat cagggggaaa atttgtccct ctcattttca
     2521 ccttgtagat cagggtccca tcctgcaggg agctgtcctg ggtcacagtc accaggcctc
     2581 cgtcctcgaa gttcatcact ctttcccact taaacccctc ggggaagctc agtttcttat
     2641 aatcgggaat gtcagctgga tgtttcacat aggccttgga gccgtacata aactgtgggc
     2701 tcaggatgtc ccaagcgaat ggcagaggcc cgccctttgt cactttcagc ttggctgtct
     2761 gggtgccctc gtagggccgt ccctctcctt ctccctcaat ttcgaactcg tggccgttca
     2821 tgcttccttc cattctcacc ttaaaccgca tgaactcttt gatcacttcc tcgcccttac
     2881 tcactgctac taccggtgcc gccactcctc cctcctcctc ctcgtcgacg aaggtctcga
     2941 tctccatgtt ctcgccggcc acgttgatgg tggttgtctt ccggatgtcg ccgtggatca
     3001 ggatgtgctc gtggatcttc actctcaggt ggtggcccag gaagccccaa gtgttcttgc
     3061 tgatcagatc caggatcagg tggccgattc cgctggtgta aggtgtgatc aggcccagtc
     3121 cctcgtgtcc ggcgatgaac atcacgggga acatcagcca agagcagaag aagacgtagg
     3181 ccatgatctt gcaaatcttc cggcagacgc ctttgggcag ggtgtggtag ctctcgatat
     3241 acaccttggc gatctggaag aagcaagtca cgccgtagaa caggccgatc atgaagaaca
     3301 ggatcttcag ggggcccttt gtaaaggcgg ctgtgatccc ccacacgatg ttgccgatgt
     3361 cggtcaccag gatggtcatg gtccgcttgc tgtactcttc gtgcagtccg gtcaggttgc
     3421 tcaggtggat caggatcacg gggcaagtca ggagccacat gctgtaccgc agccaaatca
     3481 cggctcctcc gttggtctgg tacactgtgg ctgggctgtc gacctcgtgg aacagctcga
     3541 tgaagcactt gacgagctcg atcacgcaga cgtagacctc ctcccagccg gttgtggcct
     3601 tgaagctgtg ccaaccgtag aagatgagct gcacgatagc cacgatcacg gtgaaccact
     3661 ggagtcccac tgcgatcttg tgctgcagct cggttccgtg gttgatgtgg gggaagcaac
     3721 cgtgatcggc tccggctgtt gtggcgttag aggtttctcc gtgagcgtct gctgctggtg
     3781 tcaccactgc tgcggcgctc agcaggcctc taattgtggc ttcggcgcta ggcacggctg
     3841 agataaaggc gtgggtcatt gtggcggctg tttccatggt ggcggatccg gcggcgcgcc
     3901 gaagttccta tactttctag agaataggaa cttcttgcct taacccagaa attatcactg
     3961 ttattcttta gaatggtgca aagagaagtt cctatacctt ttgaagaata ggaacttctc
     4021 cggaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     4081 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     4141 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     4201 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     4261 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     4321 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     4381 ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg
     4441 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     4501 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     4561 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gccttgtgcc
     4621 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4681 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4741 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4801 caatagcagg catgctgggg atgcggtggg ctctatggca ggaaccccta gtgatggagt
     4861 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4921 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4981 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     5041 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5101 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     5161 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5221 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     5281 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5341 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     5401 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     5461 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     5521 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     5581 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5641 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     5701 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     5761 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5821 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5881 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5941 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6001 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6061 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6121 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6181 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6241 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6301 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6361 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6421 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6481 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6541 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6601 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6661 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6721 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6781 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6841 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6901 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6961 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7021 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7081 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7141 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7201 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7261 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7321 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7381 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7441 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7501 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7561 gccttttgct cacatgt
//
