LOCUS       sequence-495319-        8606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for cocaine-gated chemogenetic anion channel.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8606)
  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 8606)
  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: "Nature. 
            2025 Aug 27. doi: 10.1038/s41586-025-09427-8."
COMMENT     Sequence Label: pCAG coca-GlyR-IRES-GFP
FEATURES             Location/Qualifiers
     source          1..8606
                     /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(5738..5757)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          5765..6781
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(5819..5841)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(6782..7059)
                     /label=chicken beta-actin promoter
     enhancer        7061..7440
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7471..7575
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7576..7644,7645..8436)
                     /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(7794..7813)
                     /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 gggggcgggg
     4321 ggaattgcgg ccgctctaga ctcgagttac tggttgtgta cgtcctccct tcgcacaatc
     4381 ttgtagatga tccaataaaa catgttgaaa atcaggaaag ccatggggaa tccgatccgg
     4441 gagattttgt cgatcttttt cgctctctga ataaagagtt tgcgcatttc ctccgggctc
     4501 ttagatggag cagggggtgg gttggttgtg ttgctattgt tggctccctt cacggaaatt
     4561 ccgtctttag cctggaggca agcaggtccc ataccatagg cgctaaagtt gaagcgtcct
     4621 tctcctgcct catcctcttt gtggtggcgt cgttttcttc tgaaccgcaa cagctcttta
     4681 tgctgtcgag acacaaaatt gacagcggcg tattccagca gtgcgctgaa gacaaacaga
     4741 aggcagacag ccatccagat atcgatggcc ttcacgtaag aaaccttggg caagctggct
     4801 cggctgcctg agctctgagt agtcattgtc agcactgtgg tgatgcccag tccgacccta
     4861 gctggagcgg cgtccatatt aatccagaag gagatccatg acaaaatcac tataagcaag
     4921 tttgggatat acatttggat cagataataa cccatgcgtc tccgcatggt cactgtgaag
     4981 gtgacatctg ggaagggctc tttgcagcat tcataaaatc gctcgcttct tttaccgggg
     5041 attcccacaa ggtcccactc cccattcggg atgtaaccag agatgtcagc ctcctgcatt
     5101 tgcagatcca ggctccattt gccgtagctc cagctcccga atttgagttt gcagtgctgg
     5161 acgtcgaagg gaaaccagcg gacgtcgata tagcatgagg acttgaagat tccagggggt
     5221 ccgtactggc agtggcctga agaattgacc aacacgttgg tgtgaaatgt tgcatcaaac
     5281 ctttcgtctg cgctgttgta cagcaggata tctggtttcc agatttggcc gtcagggaat
     5341 cttacagtct ttacacctgg gtattcgctc acattccact gcaagtagtg atcggtccag
     5401 gacatctgca accaaatatt agtggtcagc acctggttct tttcatccac gtccatgatc
     5461 tgcagcaagg agaggctgaa gtacactgtc agtggctggg aatcatttgc gaccggccgc
     5521 tccagggggt tataattctt aacaagctcc ttatacagtt tgcgctggaa ctcaccctgc
     5581 aggctaacgt gcagaaggga agcagccagg gcgagccaca cgccgcctgg agaacagcgc
     5641 atggtggcga attcaagctt gctagcccgg gtccggagat atccagctgg cgcgccgggc
     5701 ccgcggtacc gaattctttg ccaaaatgat gagacagcac aataaccagc acgttgccca
     5761 ggagctgtag gaaaaagaag aaggcatgaa catggttagc agaggctcta gagccgccgg
     5821 tcacacgcca gaagccgaac cccgccctgc cccgtccccc ccgaaggcag ccgtcccccc
     5881 gcggacagcc ccgaggctgg agagggagaa ggggacggcg gcgcggcgac gcacgaaggc
     5941 cctccccgcc catttccttc ctgccggcgc cgcaccgctt cgccccgcgc ccgctagagg
     6001 gggtgcggcg gcgcctccca gatttcggct ccgcacagat ttgggacaaa ggaagtccct
     6061 gcgccctctc gcacgattac cataaaaggc aatggctgcg gctcgccgcg cctcgacagc
     6121 cgccggcgct ccgggggccg ccgcgcccct cccccgagcc ctccccggcc cgaggcggcc
     6181 ccgccccgcc cggcaccccc acctgccgcc accccccgcc cggcacggcg agccccgcgc
     6241 cacgccccgt acggagcccc gcacccgaag ccgggccgtg ctcagcaact cggggagggg
     6301 ggtgcagggg gggttgcagc ccgaccgacg cgcccacacc ccctgctcac ccccccacgc
     6361 acacaccccg cacgcagcct ttgttcccct cgcagccccc cccgcaccgc ggggcaccgc
     6421 ccccggccgc gctcccctcg cgcacactgc ggagcgcaca aagccccgcg ccgcgcccgc
     6481 agcgctcaca gccgccgggc agcgcggagc cgcacgcggc gctccccacg cacacacaca
     6541 cgcacgcacc ccccgagccg ctccccccgc acaaagggcc ctcccggagc ccctcaaggc
     6601 tttcacgcag ccacagaaaa gaaacaagcc gtcattaaac caagcgctaa ttacagcccg
     6661 gaggagaagg gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt cagagccggg
     6721 gcgggcggcg cgaggcggcg gcggagcggg gcacggggcg aaggcagcgc gcagcgactc
     6781 ccgcccgccg cgcgcttcgc tttttatagg gccgccgccg ccgccgcctc gccataaaag
     6841 gaaactttcg gagcgcgccg ctctgattgg ctgccgccgc acctctccgc ctcgccccgc
     6901 cccgcccctc gccccgcccc gccccgcctg gcgcgcgccc cccccccccc cccgccccca
     6961 tcgctgcaca aaataattaa aaaataaata aatacaaaat tgggggtggg gagggggggg
     7021 agatggggag agtgaagcag aacgtggggc tcacctcgac catggtaata gcgatgacta
     7081 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
     7141 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     7201 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     7261 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     7321 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     7381 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     7441 gacccaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     7501 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     7561 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     7621 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     7681 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     7741 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     7801 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     7861 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     7921 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     7981 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     8041 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     8101 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     8161 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     8221 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     8281 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     8341 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     8401 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     8461 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     8521 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     8581 cccgtagaaa agatcaaagg atcttc
//
