LOCUS       sequence-273254-        6499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ChR2(H134R)-EYFP in Cre-negative cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6499)
  AUTHORS   Sermet BS, Truschow P, Feyerabend M, Mayrhofer JM, Oram TB, Yizhar 
            O, Staiger JF, Petersen CC
  TITLE     Pathway-, layer- and cell-type-specific thalamic input to mouse 
            barrel cortex.
  JOURNAL   Elife. 2019 Dec 20;8. pii: 52665. doi: 10.7554/eLife.52665.
  PUBMED    31860443
REFERENCE   2  (bases 1 to 6499)
  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"; journalName: "Elife."; 
            date: "2019-12-20"; volume: "8. pii: 52665. doi"; pages: " 
            10.7554/eLife.52665"
COMMENT     Sequence Label: pAAV-hSyn-DFO-ChR2(H134R)-EYFP-WPRE
FEATURES             Location/Qualifiers
     source          1..6499
                     /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        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      806..815
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             812..1738
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      1748..1757
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1754..1756,1757..1759,1760..2473)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1799..1820)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2060..2079)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2407..2428
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2484..2517
                     /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    2568..2601
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2626..3214
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2679..2699)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3097..3108)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3246..3722
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3445..3464)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3762..3902
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3762..3891
                     /label=AAV2 ITR
     rep_origin      3977..4432
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4064..4083)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4274..4295
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4449..4468)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4568..4590
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4628..4646)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4714..4818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4819..4887,4888..5679)
                     /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(5037..5056)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5850..6438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6339..6358
                     /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 gcggccgcac gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgatcctct agagtcgact ccggagccac catggactat ggcggcgctt tgtctgccgt
      841 cggacgcgaa cttttgttcg ttactaatcc tgtggtggtg aacgggtccg tcctggtccc
      901 tgaggatcaa tgttactgtg ccggatggat tgaatctcgc ggcacgaacg gcgctcagac
      961 cgcgtcaaat gtcctgcagt ggcttgcagc aggattcagc attttgctgc tgatgttcta
     1021 tgcctaccaa acctggaaat ctacatgcgg ctgggaggag atctatgtgt gcgccattga
     1081 aatggttaag gtgattctcg agttcttttt tgagtttaag aatccctcta tgctctacct
     1141 tgccacagga caccgggtgc agtggctgcg ctatgcagag tggctgctca cttgtcctgt
     1201 catccttatc cgcctgagca acctcaccgg cctgagcaac gactacagca ggagaaccat
     1261 gggactcctt gtctcagaca tcgggactat cgtgtggggg gctaccagcg ccatggcaac
     1321 cggctatgtt aaagtcatct tcttttgtct tggattgtgc tatggcgcga acacattttt
     1381 tcacgccgcc aaagcatata tcgagggtta tcatactgtg ccaaagggtc ggtgccgcca
     1441 ggtcgtgacc ggcatggcat ggctgttttt cgtgagctgg ggtatgttcc caattctctt
     1501 cattttgggg cccgaaggtt ttggcgtcct gagcgtctat ggctccaccg taggtcacac
     1561 gattattgat ctgatgagta aaaattgttg ggggttgttg ggacactacc tgcgcgtcct
     1621 gatccacgag cacatattga ttcacggaga tatccgcaaa accaccaaac tgaacatcgg
     1681 cggaacggag atcgaggtcg agactctcgt cgaagacgaa gccgaggccg gagccgtgcc
     1741 agcggccgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1801 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1861 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1921 gccctggccc accctcgtga ccaccttcgg ctacggcctg cagtgcttcg cccgctaccc
     1981 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2041 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     2101 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2161 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2221 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2281 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2341 gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg
     2401 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2461 gctgtacaag taagaattct agcataactt cgtataaagt atcctatacg aagttatttg
     2521 ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata acttcgtata
     2581 atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca acctctggat
     2641 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2701 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2761 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     2821 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     2881 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     2941 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3001 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc
     3061 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3121 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3181 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga
     3241 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     3301 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     3361 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     3421 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     3481 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     3541 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     3601 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     3661 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     3721 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     3781 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     3841 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     3901 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     3961 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4021 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     4081 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4141 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4201 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4261 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     4321 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     4381 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     4441 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4501 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4561 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4621 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4681 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4741 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4801 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     4861 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4921 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     4981 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5041 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5101 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5161 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5221 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5281 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5341 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5401 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5461 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5521 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5581 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5641 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5701 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5761 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5821 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5881 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5941 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6001 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6061 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6121 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6181 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6241 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6301 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6361 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6421 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6481 tggccttttg ctcacatgt
//
