LOCUS       sequence-76822-e        6657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  flexed oChIEF-citrine construct in an AAV transfer vector containing
            AAV2 ITR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6657)
  AUTHORS   Lin JY, Lin MZ, Steinbach P, Tsien RY
  TITLE     Characterization of engineered channelrhodopsin variants with 
            improved properties and kinetics.
  JOURNAL   Biophys J. 2009 Mar 4;96(5):1803-14. doi: 10.1016/j.bpj.2008.11.034.
  PUBMED    19254539
REFERENCE   2  (bases 1 to 6657)
  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: 
            "10.1016/j.bpj.2008.11"; journalName: "Biophys J"; date: "2009-03-4-
            4"; volume: "96"; issue: "5"; pages: "1803-14"
COMMENT     Sequence Label: AAV-flex-oChIEF-citrine
FEATURES             Location/Qualifiers
     source          1..6657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    228..249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(264..269,270..287,288..294)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    302..318
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     307..329
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     326..342
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     326..342
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   380..509
                     /label=AAV2 ITR
     promoter        591..1037
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(1083..1116)
                     /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(1167..1200)
                     /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(1215..1925,1926..1928,1929..1931))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1257..1278)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1606..1625
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1865..1886
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3024..3057
                     /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    3108..3141
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3166..3754
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3219..3239)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3637..3648)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3757..3773)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3758..3774)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3803..3924
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3814..3833)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3868..3887
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4086..4215
                     /label=AAV2 ITR
     primer_bind     complement(4234..4251)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4234..4250)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4243..4265)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4392..4847
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4479..4498)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4689..4710
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4873..4977
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4978..5046,5047..5838)
                     /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(5196..5215)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6009..6597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6498..6517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
       61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      181 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      241 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      301 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccagattt
      361 aattaaggcc ttaattaggc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc
      421 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      481 agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc catgctactt
      541 atctacgtag ccatgctcta ggaagatctc tgcagagggc cctgcgtatg agtgcaagtg
      601 ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccacccac
      661 tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag agggggaggg
      721 gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt
      781 cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac
      841 tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg
      901 gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc gaccatctgc
      961 gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga ggagtcgtgt
     1021 cgtgcctgag agcgcagtcg aattcaagct gatagcaagg atcctctaga gtcgactccg
     1081 gaataacttc gtataggata ctttatacga agttatgcag aatggtagct ggattgtagc
     1141 tgctattagc aatatgaaac ctcttaataa cttcgtatag catacattat acgaagttat
     1201 ggcgcgccct actacttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg
     1261 aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag ggcggactgg
     1321 tagctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc
     1381 tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat cttgaagttc
     1441 accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct gttgtagttg
     1501 tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg
     1561 atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg
     1621 tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc ggacttgaag
     1681 aagtcgtgct gcttcatgtg gtcggggtag cgggcgaagc acatcaggcc gtagccgaag
     1741 gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg
     1801 gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa cttgtggccg
     1861 tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc
     1921 ttgctcacca tctcgaggct gctctcgtac ttgccggtgc ccttgttcac ggcgccggcc
     1981 tcggcctcgt cctccaccag ggtctccacc tcgatctcgg tgccgccgat gttcagcttg
     2041 gtggtctttc tgatgtcgcc gtggatcagg atgtgctcgt ggatcagcac tctcaggtag
     2101 tggcccagca ggccccagca gttcttgctc atcaggtcga tgatggtgtg gcccacggtg
     2161 ctgccgtaca cgctcagcac gccgaagccc tcggggccca ggatgaacag gatggggaac
     2221 atgccccagc tcacgaagaa cagccaggcc atgccggtca ccacctgtct gcatctgccc
     2281 ttgggcacgg tgtggtaggc ctcgatgtac accttggcgg cgttgaagaa ggtgtagatg
     2341 ccgtagcaca ggcccatcag gaagaagatc actctcacgt agcccttgct cagggcggcg
     2401 gtggtgcccc acacgatggt gccgatgtcg ctcaccagca ggcccatggt tctcttgttg
     2461 tagtcgttgg ccaggccggt caggttgctc aggtggatca ggaccacggg gcaggtcagc
     2521 agccactcgg cgtatctcag ccacacggtc ttgttgccgt tgctgctgta gatcacggcg
     2581 ggctcgtcga actcgtggaa gtactctatg atgaacttga tcatctcgat ggtggccacg
     2641 tagatctcct cccagccgca ggtactcttc caggtctggt agccgtagaa catcaggcac
     2701 agggcgctca gggcgaaggt gatccactgc aggatgttgg cggccagctt ctcggcgttg
     2761 gtgccgttac tcttcagcca ggccaggcag aagcactggc cgttgttggg gatgcagatc
     2821 acgctgccgt tgttctccag ggtgtagctg gtctggaaca gcattctctc gtgggctctg
     2881 tggaacacgt agtcggggcc gtcctgggtg gccacgggca cggtggcgtc gctgccggtg
     2941 ctggcgccgg cgctgccggc ggccagggcc acggccaggg ccagggccag cagccagggt
     3001 cttctgctca ccatggtgct agcataactt cgtataaagt atcctatacg aagttatttg
     3061 ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata acttcgtata
     3121 atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca acctctggat
     3181 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3241 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3301 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3361 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3421 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3481 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3541 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     3601 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3661 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3721 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcgacccggg
     3781 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     3841 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3901 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3961 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     4021 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     4081 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4141 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4201 cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga
     4261 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4321 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4381 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4441 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4501 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4561 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4621 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4681 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4741 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4801 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     4861 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4921 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4981 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5041 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5101 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5161 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5221 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5281 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5341 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5401 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5461 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5521 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5581 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5641 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5701 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5761 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5821 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5881 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5941 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6001 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6061 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6121 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6181 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6241 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6301 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6361 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6421 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6481 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6541 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6601 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatg
//
