LOCUS       sequence-81542-e        8427 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Co-expresses HA tagged channelrhodopsin and hM4D (Gi).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8427)
  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 8427)
  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: CAG:: ChR2HA-2a-hM4D
FEATURES             Location/Qualifiers
     source          1..8427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1790..1799
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1796..2722
                     /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"
     CDS             2726..2752
                     /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             2726..2752
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2753..2779
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2786..2839
                     /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"
     intron          4351..4483
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4466..4485
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    4492..5080
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4545..4565)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4963..4974)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5107..5124)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5113..5224
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5451..5470)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5516..5571
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5570..5589)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5930..5946)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5943..5965)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5954..5970
                     /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)."
     promoter        complement(join(5978..5984,5985..6002,6003..6008))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6023..6044
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6103..6298
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6149..6284
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6304..6438
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6341..6360)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6395..6414
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6506..6523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6677..7265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6757..6776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7436..8227,8228..8296))
                     /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     8059..8078
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8297..8401)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg ggcgagggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcggtacc gcgggcccgg
     1741 gatccaccgg aagccacctg catgcggccg catgatcagc tggatgcatg ccaccatgga
     1801 ctatggcggc gctttgtctg ccgtcggacg cgaacttttg ttcgttacta atcctgtggt
     1861 ggtgaacggg tccgtcctgg tccctgagga tcaatgttac tgtgccggat ggattgaatc
     1921 tcgcggcacg aacggcgctc agaccgcgtc aaatgtcctg cagtggcttg cagcaggatt
     1981 cagcattttg ctgctgatgt tctatgccta ccaaacctgg aaatctacat gcggctggga
     2041 ggagatctat gtgtgcgcca ttgaaatggt taaggtgatt ctcgagttct tttttgagtt
     2101 taagaatccc tctatgctct accttgccac aggacaccgg gtgcagtggc tgcgctatgc
     2161 agagtggctg ctcacttgtc ctgtcatcct tatccgcctg agcaacctca ccggcctgag
     2221 caacgactac agcaggagaa ccatgggact ccttgtctca gacatcggga ctatcgtgtg
     2281 gggggctacc agcgccatgg caaccggcta tgttaaagtc atcttctttt gtcttggatt
     2341 gtgctatggc gcgaacacat tttttcacgc cgccaaagca tatatcgagg gttatcatac
     2401 tgtgccaaag ggtcggtgcc gccaggtcgt gaccggcatg gcatggctgt ttttcgtgag
     2461 ctggggtatg ttcccaattc tcttcatttt ggggcccgaa ggttttggcg tcctgagcgt
     2521 ctatggctcc accgtaggtc acacgattat tgatctgatg agtaaaaatt gttgggggtt
     2581 gttgggacac tacctgcgcg tcctgatcca cgagcacata ttgattcacg gagatatccg
     2641 caaaaccacc aaactgaaca tcggcggaac ggagatcgag gtcgagactc tcgtcgaaga
     2701 cgaagccgag gccggagccg tggcataccc ctacgacgtg cccgactacg cctatccata
     2761 tgatgtccca gactatgctg ctagcgaagg acgtggatct ctgctgacct gtggagatgt
     2821 ggaagagaat cccggaccat cgcgaatggc caacttcaca cctgtcaatg gcagctcggg
     2881 caatcagtcc gtgcgcctgg tcacgtcatc atcccacaat cgctatgaga cggtggaaat
     2941 ggtcttcatt gccacagtga caggctccct gagcctggtg actgtcgtgg gcaacatcct
     3001 ggtgatgctg tccatcaagg tcaacaggca gctgcagaca gtcaacaact acttcctctt
     3061 cagcctggcg tgtgctgatc tcatcatagg cgccttctcc atgaacctct acaccgtgta
     3121 catcatcaag ggctactggc ccctgggcgc cgtggtctgc gacctgtggc tggccctgga
     3181 ctgcgtggtg agcaacgcct ccgtcatgaa ccttctcatc atcagctttg accgctactt
     3241 ctgcgtcacc aagcctctca cctaccctgc ccggcgcacc accaagatgg caggcctcat
     3301 gattgctgct gcctgggtac tgtccttcgt gctctgggcg cctgccatct tgttctggca
     3361 gtttgtggtg ggtaagcgga cggtgcccga caaccagtgc ttcatccagt tcctgtccaa
     3421 cccagcagtg acctttggca cagccattgc tggcttctac ctgcctgtgg tcatcatgac
     3481 ggtgctgtac atccacatct ccctggccag tcgcagccga gtccacaagc accggcccga
     3541 gggcccgaag gagaagaaag ccaagacgct ggccttcctc aagagcccac taatgaagca
     3601 gagcgtcaag aagcccccgc ccggggaggc cgcccgggag gagctgcgca atggcaagct
     3661 ggaggaggcc cccccgccag cgctgccacc gccaccgcgc cccgtggctg ataaggacac
     3721 ttccaatgag tccagctcag gcagtgccac ccagaacacc aaggaacgcc cagccacaga
     3781 gctgtccacc acagaggcca ccacgcccgc catgcccgcc cctcccctgc agccgcgggc
     3841 cctcaaccca gcctccagat ggtccaagat ccagattgtg acgaagcaga caggcaatga
     3901 gtgtgtgaca gccattgaga ttgtgcctgc cacgccggct ggcatgcgcc ctgcggccaa
     3961 cgtggcccgc aagttcgcca gcatcgctcg caaccaggtg cgcaagaagc ggcagatggc
     4021 ggcccgggag cgcaaagtga cacgaacgat ctttgccatt ctgctagcct tcatcctcac
     4081 ctggacgccc tacaacgtca tggtcctggt gaacaccttc tgccagagct gcatccctga
     4141 cacggtgtgg tccattggct actggctctg ctacgtcaac agcaccatca accctgcctg
     4201 ctatgctctg tgcaacgcca cctttaaaaa gaccttccgg cacctgctgc tgtgccagta
     4261 tcggaacatc ggcactgcca ggtaggttat gcatcgatca cgcgtaccgg tgctcgaggt
     4321 accgatgaat agctaaggtc gaggccgcag gtaagtatca aggttacaag acaggtttaa
     4381 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca
     4441 cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcga caatcaacct
     4501 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     4561 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     4621 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     4681 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     4741 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     4801 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     4861 gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt
     4921 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     4981 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     5041 cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggaattcg agctcggtac
     5101 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     5161 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     5221 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     5281 agggggagga ttgggaagac aatagcaggc cagcttttgt tccctttagg gttaattgcg
     5341 cgcttggcgc taatcatggt catagctgtt tccactcctc aggtgcaggc tgcctatcag
     5401 aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc
     5461 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
     5521 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     5581 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
     5641 acatatgcca tatgctggct gccatgaaca aaggtggcta taaagaggtc atcagtatat
     5701 gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat
     5761 ttttttttat attttgtttg tgttattttt ttctttaaca tccctaaaat tttccttaca
     5821 tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct
     5881 tctcttatga agatccctcg acctgcagcc caagcttggc gtaatcatgg tcatagctgt
     5941 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     6001 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     6061 tgcccgcttt ccagtcggga aacctgtcgt gccagcggat ccgcatctca attagtcagc
     6121 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     6181 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     6241 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     6301 gctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     6361 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     6421 tcttatcatg tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6481 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6541 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6601 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6661 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6721 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6781 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6841 ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg
     6901 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6961 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     7021 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     7081 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     7141 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7201 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7261 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7321 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7381 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7441 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7501 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7561 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7621 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7681 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7741 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7801 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7861 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7921 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7981 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     8041 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     8101 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     8161 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     8221 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8281 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     8341 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8401 gcacatttcc ccgaaaagtg ccacctg
//
