LOCUS       sequence-390151-        7018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre recombinase dependent expression of GluClv2.0 beta subunit. 
            GluCl-beta contains a YFP tag. When co-expressed with GluClv2.0 
            alpha subunit, agonist (Ivermectin) induces neuronal silencing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7018)
  AUTHORS   Middleton SJ, Hu H, Perez-Sanchez J, Zuberi S, McGrath Williams J, 
            Weir GA, Bennett DL
  TITLE     GluCl.CreON enables selective inhibition of molecularly defined pain
            circuits.
  JOURNAL   Pain. 2023 Jun 27. doi: 10.1097/j.pain.0000000000002976.
  PUBMED    37366588
REFERENCE   2  (bases 1 to 7018)
  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: "Pain. 2023 
            Jun 27. doi: 10.1097/j.pain.0000000000002976."
COMMENT     Sequence Label: pAAV-sCAG-GluCl-beta.CreON
FEATURES             Location/Qualifiers
     source          1..7018
                     /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..3176
                     /label=chicken beta-actin promoter
     primer_bind     3455..3477
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3539..3558
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(3615..3648)
                     /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(3699..3732)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(3942..4652,4653..4655,4656..4658))
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3984..4005)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4333..4352
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4592..4613
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    5778..5811
                     /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    5862..5895
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5920..6508
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5973..5993)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6391..6402)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6551..6672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6562..6581)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6616..6635
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6834..6963
                     /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 cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg
     3061 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
     3121 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
     3181 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
     3241 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
     3301 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
     3361 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
     3421 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     3481 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     3541 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcg ggatcctcta
     3601 gagtcgactc cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag
     3661 ctggattgta gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt
     3721 atacgaagtt atggcgcgcc ctacaccagg gactcggggg tgctggcgtt agcgctcatg
     3781 atcaggcagg acacccagta gatcacgttg aaggccagga aggccagggg caccacgaag
     3841 cgggcgtaga agtcgatctt ggcgggcagg tagtcgggct tggtgaagta gcgtctgact
     3901 ttgtcgcgga aggtctctct ggcctcgaag gtgtggcacg tcttgtacag ctcgtccatg
     3961 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
     4021 ttggggtctt tgctcagctt ggactggtag ctcaggtagt ggttgtcggg cagcagcacg
     4081 gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc
     4141 tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg
     4201 atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc
     4261 tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc
     4321 acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg
     4381 tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg
     4441 gcgaagcact gcaggccgta gccgaaggtg gtcacgaggg tgggccaggg cacgggcagc
     4501 ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc
     4561 tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc
     4621 accaccccgg tgaacagctc ctcgcccttg ctcaccatcg tgcagggggg ctggtacacc
     4681 tcggcgtcca ccatctccag cttctcgcgg cgtctctggg ccttgcgggc cgccttctcg
     4741 gcggcggcgt cgttctggcg cacggagtct tggtaggaga cgaaggcgta ctccagcagg
     4801 gcgccgaaca cgaaggtctg gcaggcgccc agccacacgt ccacgacttt cacgtaggac
     4861 acggggggca gcttggcgtt gatggcgctc tgcatggtgg tcatggtcag cagggtggtc
     4921 acgcccaggg ccacgcggcc ggcggtgctg tgcaggtcga tccagaagct gacccagctc
     4981 acgatcacga tcatggtggt gggggcgtac agctgcacca ggtagtagct gaactggcgc
     5041 ttgaacagca gctgcatgcg caggcagccg tacgagccag tgttggtgtg ggaggtgcag
     5101 tcggcgttgg tggtgtagtt cttcaggatg aagttgggca ggtcggagcc cacgccgggc
     5161 ttcagctgca cgggggtgct ggggtcccac tcatacatga tgtcgttcat ggtgtgggcg
     5221 aaggacacca ggtcgaagtt gcagctctgg tagtccaggg ggtacagctg caggcgcatg
     5281 gggcaggagc tcgtcaggct gatgcggctg ctgtacagga tcttgccgtc ggggtagatg
     5341 cgcaggaaca tgttctccat gtcgatcagg tggcggtggg cggccttctc ggtggggaag
     5401 aaggtgtcgg gtatccacag ggacttcttg acgtggggca cggtcaggaa ggcggggggg
     5461 ttgtagaagc ccaggttctc gtaggccagg cgggggtcga tccactgctc gcggaaggtc
     5521 agctggatgg agtactccat gttcaccacg tcgatcttgc tcagcattct gatcatgatg
     5581 ttcactctca cgttcacggc gccctcggtg ctgctgttgg cggggggggg gcgcacgcgc
     5641 atgtcgtagt tcttcagcag ggcattcagg atctcctgct cgctctgcat gctgtactcg
     5701 ccgttggtca ccacgggcat cagcagcagc agcagcagga tcgagaagct ggagggggtg
     5761 gccatggtgg ggctagcata acttcgtata aagtatccta tacgaagtta tttgccttaa
     5821 cccagaaatt atcactgtta ttctttagaa tggtgcaaag aataacttcg tataatgtat
     5881 gctatacgaa gttatgaatt cgatatcaag cttatcgata atcaacctct ggattacaaa
     5941 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     6001 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     6061 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     6121 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     6181 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     6241 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     6301 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     6361 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     6421 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     6481 cggatctccc tttgggccgc ctccccgcat cgataccgtc gacccgggcg gccgcttcga
     6541 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     6601 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     6661 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     6721 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt
     6781 cctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc
     6841 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     6901 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     6961 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgct
//
