LOCUS       sequence-487705-        8708 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Expression vector for cocaine-gated chemogenetic cation channel.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8708)
  AUTHORS   Gomez JL, Magnus CJ, Bonaventura J, Solis O, Curry FP, Levinstein 
            MR, Budinich RC, Carlton ML, Ventriglia EN, Lam S, Wang L, 
            Schoenborn I, Dunne W, Michaelides M, Sternson SM
  TITLE     Cocaine chemogenetics blunts drug-seeking by synthetic physiology.
  JOURNAL   Nature. 2025 Aug 27. doi: 10.1038/s41586-025-09427-8.
  PUBMED    40866713
REFERENCE   2  (bases 1 to 8708)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2025 Aug 27. doi: 10.1038/s41586-025-09427-8."
COMMENT     Sequence Label: pCAG coca-5HT3-IRES-GFP
FEATURES             Location/Qualifiers
     source          1..8708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    828..962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(852..871)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     906..925
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(968..1163)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      982..1117
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    1222..1243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1258..1263,1264..1281,1282..1288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1296..1312
                     /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     1320..1336
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1679..1698
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1697..1752
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1798..1817
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     primer_bind     1919..1935
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1954..1974
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1956..1974
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    2070..2181
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2170..2187
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     misc_feature    2214..2802
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             2320..2331
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2729..2749
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(2809..2828)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          2811..2943
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             complement(join(2988..3701,3702..3704))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3033..3054)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3382..3401
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3641..3662
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    join(3717..3725,3726..3728,3729..4302)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     4118..4135
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(5840..5859)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          5867..6883
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(5921..5943)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        7163..7542
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7573..7677
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7678..7746,7747..8538)
                     /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(7896..7915)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcggat ccagacatga
      841 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
      901 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      961 ttagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     1021 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     1081 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     1141 tggttgctga ctaattgaga tgcggatccg ctggcacgac aggtttcccg actggaaagc
     1201 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     1261 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     1321 aggaaacagc tatgaccatg attacgccaa gcttgggctg caggtcgagg gatctccata
     1381 agagaagagg gacagctatg actgggagta gtcaggagag gaggaaaaat ctggctagta
     1441 aaacatgtaa ggaaaatttt agggatgtta aagaaaaaaa taacacaaaa caaaatataa
     1501 aaaaaatcta acctcaagtc aaggcttttc tatggaataa ggaatggaca gcagggggct
     1561 gtttcatata ctgatgacct ctttatagcc aacctttgtt catggcagcc agcatatggg
     1621 catatgttgc caaactctaa accaaatact cattctgatg ttttaaatga tttgccctcc
     1681 catatgtcct tccgagtgag agacacaaaa aattccaaca cactattgca atgaaaataa
     1741 atttccttta ttagccagaa gtcagatgct caaggggctt catgatgtcc ccataatttt
     1801 tggcagaggg aaaaagatct cagtggtatt tgtgagccag ggcattggcc acaccagcca
     1861 ccaccttctg ataggcagcc tgcacctgag gagtgaatta gcggataaca atttcacaca
     1921 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
     1981 aagctgggct attgtcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca
     2041 gaatagaatg acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga
     2101 cagtgggagt ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg
     2161 gctggcaact agaaggcaca gtcgaggctg atcgtaccga gctcgaattc caggcgggga
     2221 ggcggcccaa agggagatcc gactcgtctg agggcgaagg cgaagacgcg gaagaggccg
     2281 cagagccggc agcaggccgc gggaaggaag gtccgctgga ttgagggccg aagggacgta
     2341 gcagaaggac gtcccgcgca gaatccaggt ggcaacacag gcgagcagcc atggaaagga
     2401 cgtcagcttc cccgacaaca ccacggaatt gtcagtgccc aacagccgag cccctgtcca
     2461 gcagcgggca aggcaggcgg cgatgagttc cgccgtggca atagggaggg ggaaagcgaa
     2521 agtcccggaa aggagctgac aggtggtggc aatgccccaa ccagtggggg ttgcgtcagc
     2581 aaacacagtg cacaccacgc cacgttgcct gacaacgggc cacaactcct cataaagaga
     2641 cagcaaccag gatttataca aggaggagaa aatgaaagcc atacgggaag caatagcatg
     2701 atacaaaggc attaaagcag cgtatccaca tagcgtaaaa ggagcaacat agttaagaat
     2761 accagtcaat ctttcacaaa ttttgtaatc cagaggttga ttgtcgacac ctgtggagag
     2821 aaaggcaaag tggatgtcag taagaccaat aggtgcctat cagaaacgca agagtcttct
     2881 ctgtctcgac aagcccagtt tctattggtc tccttaaacc tgtcttgtaa ccttgatact
     2941 tacctgcggc ctcgacctta gctattcatc ggtacctcga ctctagatta cttgtacagc
     3001 tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg
     3061 cgcttctcgt tggggtcttt gctcagcttg gactgggtgc tcaggtagtg gttgtcgggc
     3121 agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg
     3181 ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt cttctgcttg
     3241 tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg ccccaggatg
     3301 ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc
     3361 tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc
     3421 tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg
     3481 gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt gggccagggc
     3541 acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg
     3601 ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc
     3661 aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccggatc ccccatggtt
     3721 gtggccatat tatcatcgtg tttttcaaag gaaaaccacg tccccgtggt tcggggggcc
     3781 tagacgtttt tttaacctcg actaaacaca tgtaaagcat gtgcaccgag gccccagatc
     3841 agatcccata caatggggta ccttctgggc atccttcagc cccttgttga atacgcttag
     3901 gagagccatt tgactctttc cacaactatc caactcacaa cgtggcactg gggttgtgcc
     3961 gcctttgcag gtgtatctta tacacgtggc ttttggccgc agaggcacct gtcgccaggt
     4021 ggggggttcc gctgcctgca aagggtcgct acagacgttg tttgtcttca agaagcttcc
     4081 agaggaactg cttccttcac gacattcaac agaccttgca ttcctttggc gagaggggaa
     4141 agacccctag gaatgctcgt caagaagaca gggccaggtt tccgggccct cacattgcca
     4201 aaagacggca atatggtgga aaataacata tagacaaacg cacaccggcc ttattccaag
     4261 cggcttcggc cagtaacgtt aggggggggg gagggagagg gggggggggg gggggggggg
     4321 ggaattgcgg ccgcctaact gtagtgccat atggaccaga gtgtcaccag agtaatggaa
     4381 taagccagca ccgccagcaa gtagatccga aacagcagcc tgtcaaggac gtagcccact
     4441 cgcagccagt cgcgagcgac ttcgcgcatc tcgtcccgct tctccagaaa atgtcggatg
     4501 cttgacagtt cctggagcag cccccggaca gcgagagagg cttctcgggg aggaggaagg
     4561 ggtgatcccc gcccccgtgg ggtcttttcc aaatcttgtg gtcccccaac atgggagcag
     4621 tgattcccca tagcaggcaa cagatcagac ccactacaat cgtctgtctt gttggcttga
     4681 aaggttgcag gtggccggtg ggccataggt tgctcgccaa gacacagaat ccatgcaatt
     4741 ctgtccagca caaggtgtcg caaccagtct ggcacgggtc tttgcaggtc ctgcttatgt
     4801 accagccgta caatgaagat agtttccgcc agacttatca ccaggagtgc catacacacg
     4861 acgaaataca ctcctatcag cggagtgccg attgtggcag gaagggtatc ggacacgatg
     4921 atcagaaata cagaatagcc gagcaacaag gtaatcttaa aggagaccct ctccccgcta
     4981 tcaggtggga gacaaaatcc cacgatgtcc acgaccatga gaaaaatact ggggagcaac
     5041 aaactaacgg cgtagaacaa tggccttctt ctgatgataa ctgtgaatgt tacgtcaggg
     5101 aagggttcct tgcagcattc aaaaaatcgc tcgcttcttt taccggggat tcccacaagg
     5161 tcccactccc cattcgggat gtaaccagag atgtcagcct cctgcatttg cagatccagg
     5221 ctccatttgc cgtagctcca gctcccgaat ttgagtttgc agtgctggac gtcgaaggga
     5281 aaccagcgga cgtcgatata gcatgaggac ttgaagattc cagggggtcc gtactggcag
     5341 tggcctgaag aattgaccaa cacgttggtg tgaaatgttg catcaaacct ttcgtctgcg
     5401 ctgttgtaca gcaggatatc tggtttccag atttggccgt cagggaatct tacagtcttt
     5461 acacctgggt attcgctcac attccactgc aagtagtgat cggtccagga catctgcaac
     5521 caaatattag tggtcagcac ctggttcttt tcatccacgt ccatgatctg cagcaaggag
     5581 aggctgaagt acactgtcag tggctgggaa tcatttgcga ccggccgctc cagggggtta
     5641 taattcttaa caagctcctt atacagtttg cgctggaact caccctgcag gctaacgtgc
     5701 agaagggaag cagccagggc gagccacacg ccgcctggag aacagcgcat ggtggcgaat
     5761 tcgctagccc gggtccggag atatccagct ggcgcgccgg gcccgcggta ccgaattctt
     5821 tgccaaaatg atgagacagc acaataacca gcacgttgcc caggagctgt aggaaaaaga
     5881 agaaggcatg aacatggtta gcagaggctc tagagccgcc ggtcacacgc cagaagccga
     5941 accccgccct gccccgtccc ccccgaaggc agccgtcccc ccgcggacag ccccgaggct
     6001 ggagagggag aaggggacgg cggcgcggcg acgcacgaag gccctccccg cccatttcct
     6061 tcctgccggc gccgcaccgc ttcgccccgc gcccgctaga gggggtgcgg cggcgcctcc
     6121 cagatttcgg ctccgcacag atttgggaca aaggaagtcc ctgcgccctc tcgcacgatt
     6181 accataaaag gcaatggctg cggctcgccg cgcctcgaca gccgccggcg ctccgggggc
     6241 cgccgcgccc cycccccgag cccyccccgg cccgrggcgg ccccgccccg cccggcaccc
     6301 ccacctgccg ccaccccccg cccggcacgg cgagccccgc gccacgcccc gtacggagcc
     6361 ccgcacccga agccgggccg tgctcagcaa ctcggggagg ggggtgcagg gggggttgca
     6421 gcccgaccga cgcgcccaca ccccctgctc acccccccac gcacacaccc cgcacgcagc
     6481 ctttgttccc ctcgcagccc cccccgcacc gcggggcacc gcccccggcc gcgctcccct
     6541 cgcgcacact gcggagcgca caaagccccg cgccgcgccc gcagcgctca cagccgccgg
     6601 gcagcgcgga gccgcacgcg gcgctcccca cgcacacaca cacgcacgca ccccccgagc
     6661 cgctcccccc gcacaaaggg ccctcccgga gcccctcaag gctttcacgc agccacagaa
     6721 aagaaacaag ccgtcattaa accaagcgct aattacagcc cggaggagaa gggccgtccc
     6781 gcccgctcac ctgtgggagt aacgcggtca gtcagagccg gggcgggcgg cgcgaggcgg
     6841 cggcggagcg gggcacgggg cgaaggcagc gcgcagcgac tcccgcccgc cgcgcgcttc
     6901 gctttttata gggccgccgc cgccgccgcc tcgccataaa aggaaacttt cggagcgcgc
     6961 cgctctgatt ggctgccgcc gcmccycycc sccycscccc sccccccccc cccccccccc
     7021 cccccccccc cggcgcgcgc cccccccccc cccccgcccc catcgctgca caaaataatt
     7081 aaaaaataaa taaatacaaa attgggggtg gggagggggg ggagatgggg agagtgaagc
     7141 agaacgtggg gctcacctcg accatggtaa tagcgatgac taatacgtag atgtactgcc
     7201 aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt
     7261 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg
     7321 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta
     7381 tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg
     7441 ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc
     7501 ccgtaattga ttactattaa taactagtca ataatcaatg tcgacccagg tggcactttt
     7561 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     7621 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     7681 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     7741 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     7801 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     7861 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     7921 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     7981 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     8041 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     8101 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     8161 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     8221 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     8281 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     8341 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     8401 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     8461 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     8521 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     8581 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     8641 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     8701 ggatcttc
//
