LOCUS       sequence-142412-        7396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of ChR2 and PSAM-GlyR (L141F,Y115F) 
            neuronal inhibitor, with IRES-EGFP marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7396)
  AUTHORS   Magnus CJ, Lee PH, Atasoy D, Su HH, Looger LL, Sternson SM
  TITLE     Chemical and genetic engineering of selective ion channel-ligand 
            interactions.
  JOURNAL   Science. 2011 Sep 2;333(6047):1292-6.
  PUBMED    21885782
REFERENCE   2  (bases 1 to 7396)
  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: "Science."; 
            date: "2011-09-2"; volume: "333(6047)"; pages: "1292-6"
COMMENT     Sequence Label: rAAV-CAG::FLEX-rev:ChR2HA:2a:PSAML141F,Y115F:GlyR
FEATURES             Location/Qualifiers
     source          1..7396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   13..142
                     /label=AAV2 ITR
     promoter        453..730
                     /label=chicken beta-actin promoter
     primer_bind     1009..1031
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1093..1112
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1147..1180
                     /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    1243..1276
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2599..2652)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(2659..2685)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2686..2712)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             complement(2686..2712)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2716..3642)
                     /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      3639..3648
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3673..3706)
                     /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(3777..3810)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3852..4440
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3905..3925)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4323..4334)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4483..4604
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4494..4513)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4548..4567
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4766..4895
                     /label=AAV2 ITR
     rep_origin      complement(5132..5720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5212..5231)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5891..6682,6683..6751))
                     /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     6514..6533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6752..6856)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6882..7337)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7019..7040)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7231..7250
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctattacgcc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
       61 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      121 aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctacg
      181 tagccatgct ctaggaagag taccattgac gtcaataatg acgtatgttc ccatagtaac
      241 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      301 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      361 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      421 catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac
      481 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt
      541 ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg
      601 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct
      661 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc
      721 gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc
      781 gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg
      841 cccttctcct ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg
      901 gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg
      961 gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct
     1021 ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     1081 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgg
     1141 atccccataa cttcgtataa agtatcctat acgaagttat atcaaaatag gaagaccaat
     1201 gcttcaccat cgacccgaat tgccaagcat caccatcgac ccataacttc gtataatgta
     1261 tgctatacga agttatacta gcgcgtgata tcgaacttac tggttgtgta cgtcctccct
     1321 tcgcacaatc ttgtagatga tccaataaaa catgttgaaa atcaggaaag ccatggggaa
     1381 tccgatccgg gagattttgt cgatcttttt cgctctctga ataaagagtt tgcgcatttc
     1441 ctccgggctc ttagatggag cagggggtgg gttggttgtg ttgctattgt tggctccctt
     1501 cacggaaatt ccgtctttag cctggaggca agcaggtccc ataccatagg cgctaaagtt
     1561 gaagcgtcct tctcctgcct catcctcttt gtggtggcgt cgttttcttc tgaaccgcaa
     1621 cagctcttta tgctgtcgag acacaaaatt gacagcggcg tattccagca gtgcgctgaa
     1681 gacaaacaga aggcagacag ccatccagat atcgatggcc ttcacgtaag aaaccttggg
     1741 caagctggct cggctgcctg agctctgagt agtcattgtc agcactgtgg tgatgcccag
     1801 tccgacccta gctggagcgg cgtccatatt aatccagaag gagatccatg acaaaatcac
     1861 tataagcaag cttgggatat acatttggat cagataataa cccatgcgtc tccgcatggt
     1921 cactgtgaag gtgacatctg ggtagggctc tttgcagcat tcataaaatc gctcgcttct
     1981 tttaccgggg attcccacaa ggtcccactc cccattcggg atgtaaccag agatgtcagc
     2041 ctcctgcatt tgcagatcca ggctccatcc gccgtagctc cagctcccga atttgagttt
     2101 gcagtgctgg acgtcgaagg gaaaccagcg gacgtcgata tagcatgagg acttgaagat
     2161 tccagggggg aagtactggc agtggcctga agaattgacc aacacgttgg tgtgaaatgt
     2221 tgcatcaaac ctttcgtctg cgctgttgaa cagcaggata tctggtttcc agatttggcc
     2281 gtcagggaat cttacagtct ttacacctgg gtattcgctc acattccact gcaagtagtg
     2341 atcggtccag gacatctgca accaaatatt agtggtcagc acctggttct tttcatccac
     2401 gtccatgatc tgcagcaagg agaggctgaa gtacactgtc agtggctggg aatcatttgc
     2461 gaccggccgc tccagggggt tataattctt aacaagctcc ttatacagtt tgcgctggaa
     2521 ctcaccctgc aggctaacgt gcagaaggga agcagccagg gcgagccaca cgccgcctgg
     2581 agaacagcgc attcgcgatg gtccgggatt ctcttccaca tctccacagg tcagcagaga
     2641 tccacgtcct tcgctagcag catagtctgg gacatcatat ggataggcgt agtcgggcac
     2701 gtcgtagggg tatgccacgg ctccggcctc ggcttcgtct tcgacgagag tctcgacctc
     2761 gatctccgtt ccgccgatgt tcagtttggt ggttttgcgg atatctccgt gaatcaatat
     2821 gtgctcgtgg atcaggacgc gcaggtagtg tcccaacaac ccccaacaat ttttactcat
     2881 cagatcaata atcgtgtgac ctacggtgga gccatagacg ctcaggacgc caaaaccttc
     2941 gggccccaaa atgaagagaa ttgggaacat accccagctc acgaaaaaca gccatgccat
     3001 gccggtcacg acctggcggc accgaccctt tggcacagta tgataaccct cgatatatgc
     3061 tttggcggcg tgaaaaaatg tgttcgcgcc atagcacaat ccaagacaaa agaagatgac
     3121 tttaacatag ccggttgcca tggcgctggt agccccccac acgatagtcc cgatgtctga
     3181 gacaaggagt cccatggttc tcctgctgta gtcgttgctc aggccggtga ggttgctcag
     3241 gcggataagg atgacaggac aagtgagcag ccactctgca tagcgcagcc actgcacccg
     3301 gtgtcctgtg gcaaggtaga gcatagaggg attcttaaac tcaaaaaaga actcgagaat
     3361 caccttaacc atttcaatgg cgcacacata gatctcctcc cagccgcatg tagatttcca
     3421 ggtttggtag gcatagaaca tcagcagcaa aatgctgaat cctgctgcaa gccactgcag
     3481 gacatttgac gcggtctgag cgccgttcgt gccgcgagat tcaatccatc cggcacagta
     3541 acattgatcc tcagggacca ggacggaccc gttcaccacc acaggattag taacgaacaa
     3601 aagttcgcgt ccgacggcag acaaagcgcc gccatagtcc atggtggcgg atccgtttaa
     3661 acactagcta gtataacttc gtataggata ctttatacga agttatcatt gggattcttc
     3721 ctattttgat ccaagcatca ccatcgaccc tctagtccag atctcaccat cgacccataa
     3781 cttcgtatag catacattat acgaagttat gtccctcgaa gaggttcgaa ttcgatatca
     3841 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3901 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3961 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     4021 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     4081 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     4141 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     4201 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     4261 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     4321 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     4381 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     4441 atcgataccg tcgacccggg cggccgcttc gagcagacat gataagatac attgatgagt
     4501 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     4561 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     4621 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc
     4681 tctacaaatg tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag
     4741 catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct
     4801 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4861 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg
     4921 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4981 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5041 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5101 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5161 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5221 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5281 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5341 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5401 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5461 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5521 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5581 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5641 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5701 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5761 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5821 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5881 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5941 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6001 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6061 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6121 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6181 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6241 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6301 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6361 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6421 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6481 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6541 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6601 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6661 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6721 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6781 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6841 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     6901 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     6961 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     7021 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     7081 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     7141 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     7201 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     7261 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     7321 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     7381 ggtgcgggcc tcttcg
//
