LOCUS       sequence-142391-        7490 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Co-expresses HA tagged channelrhodopsin and hM4D (Gi) from a 
            Cre-dependent virus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7490)
  AUTHORS   Stachniak TJ, Ghosh A, Sternson SM
  TITLE     Chemogenetic synaptic silencing of neural circuits localizes a 
            hypothalamus-->midbrain pathway for feeding behavior.
  JOURNAL   Neuron. 2014 May 21;82(4):797-808. doi: 
            10.1016/j.neuron.2014.04.008. Epub 2014 Apr 24.
  PUBMED    24768300
REFERENCE   2  (bases 1 to 7490)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neuron.2014.04.008"; journalName: "Neuron"; date: 
            "2014-05-21- 21"; volume: "82"; issue: "4"; pages: "797-808"
COMMENT     Sequence Label: AAV-CAG::FLEX-rev:: ChR2HA-2a-hM4D
FEATURES             Location/Qualifiers
     source          1..7490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     promoter        2899..3155
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     primer_bind     3434..3456
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3518..3537
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    3572..3605
                     /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    3668..3701
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(5157..5210)
                     /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(5217..5243)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(5244..5270)
                     /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(5244..5270)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(5274..6200)
                     /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      6197..6206
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(6213..6246)
                     /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(6317..6350)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6392..6980
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6445..6465)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6863..6874)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7023..7144
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7034..7053)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7088..7107
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   7306..7435
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2701 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2761 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2821 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2881 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
     2941 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
     3001 gggcgggggg gggggggggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg
     3061 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
     3121 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
     3181 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     3241 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt
     3301 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
     3361 ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     3421 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     3481 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     3541 tctcatcatt ttggcaaaga attggatccc cataacttcg tataaagtat cctatacgaa
     3601 gttatatcaa aataggaaga ccaatgcttc accatcgacc cgaattgcca agcatcacca
     3661 tcgacccata acttcgtata atgtatgcta tacgaagtta tactagtaac ctacctggca
     3721 gtgccgatgt tccgatactg gcacagcagc aggtgccgga aggtcttttt aaaggtggcg
     3781 ttgcacagag catagcaggc agggttgatg gtgctgttga cgtagcagag ccagtagcca
     3841 atggaccaca ccgtgtcagg gatgcagctc tggcagaagg tgttcaccag gaccatgacg
     3901 ttgtagggcg tccaggtgag gatgaaggct agcagaatgg caaagatcgt tcgtgtcact
     3961 ttgcgctccc gggccgccat ctgccgcttc ttgcgcacct ggttgcgagc gatgctggcg
     4021 aacttgcggg ccacgttggc cgcagggcgc atgccagccg gcgtggcagg cacaatctca
     4081 atggctgtca cacactcatt gcctgtctgc ttcgtcacaa tctggatctt ggaccatctg
     4141 gaggctgggt tgagggcccg cggctgcagg ggaggggcgg gcatggcggg cgtggtggcc
     4201 tctgtggtgg acagctctgt ggctgggcgt tccttggtgt tctgggtggc actgcctgag
     4261 ctggactcat tggaagtgtc cttatcagcc acggggcgcg gtggcggtgg cagcgctggc
     4321 gggggggcct cctccagctt gccattgcgc agctcctccc gggcggcctc cccgggcggg
     4381 ggcttcttga cgctctgctt cattagtggg ctcttgagga aggccagcgt cttggctttc
     4441 ttctccttcg ggccctcggg ccggtgcttg tggactcggc tgcgactggc cagggagatg
     4501 tggatgtaca gcaccgtcat gatgaccaca ggcaggtaga agccagcaat ggctgtgcca
     4561 aaggtcactg ctgggttgga caggaactgg atgaagcact ggttgtcggg caccgtccgc
     4621 ttacccacca caaactgcca gaacaagatg gcaggcgccc agagcacgaa ggacagtacc
     4681 caggcagcag caatcatgag gcctgccatc ttggtggtgc gccgggcagg gtaggtgaga
     4741 ggcttggtga cgcagaagta gcggtcaaag ctgatgatga gaaggttcat gacggaggcg
     4801 ttgctcacca cgcagtccag ggccagccac aggtcgcaga ccacggcgcc caggggccag
     4861 tagcccttga tgatgtacac ggtgtagagg ttcatggaga aggcgcctat gatgagatca
     4921 gcacacgcca ggctgaagag gaagtagttg ttgactgtct gcagctgcct gttgaccttg
     4981 atggacagca tcaccaggat gttgcccacg acagtcacca ggctcaggga gcctgtcact
     5041 gtggcaatga agaccatttc caccgtctca tagcgattgt gggatgatga cgtgaccagg
     5101 cgcacggact gattgcccga gctgccattg acaggtgtga agttggccat tcgcgatggt
     5161 ccgggattct cttccacatc tccacaggtc agcagagatc cacgtccttc gctagcagca
     5221 tagtctggga catcatatgg ataggcgtag tcgggcacgt cgtaggggta tgccacggct
     5281 ccggcctcgg cttcgtcttc gacgagagtc tcgacctcga tctccgttcc gccgatgttc
     5341 agtttggtgg ttttgcggat atctccgtga atcaatatgt gctcgtggat caggacgcgc
     5401 aggtagtgtc ccaacaaccc ccaacaattt ttactcatca gatcaataat cgtgtgacct
     5461 acggtggagc catagacgct caggacgcca aaaccttcgg gccccaaaat gaagagaatt
     5521 gggaacatac cccagctcac gaaaaacagc catgccatgc cggtcacgac ctggcggcac
     5581 cgaccctttg gcacagtatg ataaccctcg atatatgctt tggcggcgtg aaaaaatgtg
     5641 ttcgcgccat agcacaatcc aagacaaaag aagatgactt taacatagcc ggttgccatg
     5701 gcgctggtag ccccccacac gatagtcccg atgtctgaga caaggagtcc catggttctc
     5761 ctgctgtagt cgttgctcag gccggtgagg ttgctcaggc ggataaggat gacaggacaa
     5821 gtgagcagcc actctgcata gcgcagccac tgcacccggt gtcctgtggc aaggtagagc
     5881 atagagggat tcttaaactc aaaaaagaac tcgagaatca ccttaaccat ttcaatggcg
     5941 cacacataga tctcctccca gccgcatgta gatttccagg tttggtaggc atagaacatc
     6001 agcagcaaaa tgctgaatcc tgctgcaagc cactgcagga catttgacgc ggtctgagcg
     6061 ccgttcgtgc cgcgagattc aatccatccg gcacagtaac attgatcctc agggaccagg
     6121 acggacccgt tcaccaccac aggattagta acgaacaaaa gttcgcgtcc gacggcagac
     6181 aaagcgccgc catagtccat ggtggcacta gtataacttc gtataggata ctttatacga
     6241 agttatcatt gggattcttc ctattttgat ccaagcatca ccatcgaccc tctagtccag
     6301 atctcaccat cgacccataa cttcgtatag catacattat acgaagttat gtccctcgaa
     6361 gaggttcgaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     6421 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     6481 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     6541 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     6601 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     6661 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     6721 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     6781 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     6841 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     6901 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     6961 cctttgggcc gcctccccgc atcgataccg tcgacccggg cggccgcttc gagcagacat
     7021 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     7081 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     7141 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
     7201 tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc ttcctagagc
     7261 atggctacgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga
     7321 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     7381 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca
     7441 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
//
