LOCUS       sequence-390150-        7102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre recombinase dependent expression of GluClv2.0 alpha subunit. 
            GluCl-alpha contains a Cerulean tag. When co-expressed with 
            GluClv2.0 beta subunit, agonist (Ivermectin) induces neuronal 
            silencing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7102)
  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 7102)
  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-alpha.CreON
FEATURES             Location/Qualifiers
     source          1..7102
                     /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(3894..4604,4605..4607,4608..4610))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3936..3957)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4285..4304
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4544..4565
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    5862..5895
                     /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    5946..5979
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6004..6592
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6057..6077)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6475..6486)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6635..6756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6646..6665)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6700..6719
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6918..7047
                     /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 tcagaacaga acgttctgct ggccgaagcg gctccagtac
     3781 aggatgttga acacgaagaa cagcacgggg aacagggcgc ggctgatcag gtccacgcgc
     3841 ttgctgatgt cgttccactc ggtcttggtc tccagcaggt tcaggaagga attcttgtac
     3901 agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga
     3961 tcgcgcttct cgttggggtc tttgctcagc ttggactggg tgctcaggta gtggttgtcg
     4021 ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc
     4081 acgctgccgt cctcgatgtt gtggcggatc ttgaagttgg ccttgatgcc gttcttctgc
     4141 ttgtcggcgg tgatatagac gttgtcgctg atggcgttgt actccagctt gtgccccagg
     4201 atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg
     4261 ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg
     4321 cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg
     4381 tcggggtagc gggcgaagca ctgcacgccc caggtcaggg tggtcacgag ggtgggccag
     4441 ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca
     4501 tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg
     4561 accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggaattcatc
     4621 tgccggcggt tcaggggcac ggtgcggact ttctcctcct ggttgaacac cttggtgggg
     4681 acgtcgttgt gcaggttctg cagcaggggg atctcggcct tctcgcgctc ggtgcgggcc
     4741 ttgcgctcca cgccctgctt gttcgcgatg tggttaacca gggcgaactc cagcagggcg
     4801 cagaagatga aggtcatgca ggcgccgatc cacacgtcga tggccttgat gtagctcacg
     4861 gggggcagct ggctgttgat gccagcggac tgagcggtca tggtgaacag ggtggtcacg
     4921 cccagggtga cgcgggcggg gatggcggtg cggtcgaacc agaaggacac ccaggacacg
     4981 atcacgagca tgcagctggg gatgtacagt tgcagcaggt agaaggagaa ctcgcgcttc
     5041 agctggatgg tggtcctcag gcagctgtag atgccggtgt tggtgacgct ggtgcagtag
     5101 gtggtgctgg tgttggtcag ctggaagctg ggcaggctgg aggacaggcc gaccttcagc
     5161 tgcagggggc tgtgctcctt ccacagatac tcgatgtcct tggtggtgta ggcgtaggag
     5221 gccaggtcga tgctgcactg ctgcacgtcc atcgggtagt actgcaggta catggggcag
     5281 ctcagcacca ggctgatgcg gacggagtac aggacggtgc cgtcgttgtg gatgcggatc
     5341 aggacgttgg gcttgtcgat ggtgtgcttg taagcctgct tctcgttggg gaagaaggtg
     5401 tcgggcatcc agatctggtg gcccacggtc aggatgacga agtcgggctg gccgtcgccc
     5461 ttgacgccgt aggacaggcg cttgtcgatc cagctctcgc gcagggtcag ctgggcggag
     5521 tactccatgt tcaccacgtc gatcttgctg atggtgcgca gcagcatgtt cacgctcacc
     5581 accacggggc cgccgttgtc ggtggggggg cgcacgcgga agtcgtagcc gctggtgaac
     5641 aggtgggcca ggatcttgct gtcggaggtc tggggctgct cgatggggat gtggatgggg
     5701 gaggtcaggc gagccaggga ctccagggtg gtagtgccgt tgtcgttgtc gtcctcgaag
     5761 atgtcctggc tcttggtgcg ggcctgctgg gcctggatcc ccaggatcag ggaagcgatg
     5821 atcagcttgc ccacgatcca cgtggccatg gtggggctag cataacttcg tataaagtat
     5881 cctatacgaa gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc
     5941 aaagaataac ttcgtataat gtatgctata cgaagttatg aattcgatat caagcttatc
     6001 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     6061 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     6121 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     6181 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     6241 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     6301 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     6361 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     6421 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     6481 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     6541 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     6601 cgtcgacccg ggcggccgct tcgagcagac atgataagat acattgatga gtttggacaa
     6661 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     6721 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     6781 atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa
     6841 tgtggtaaaa tcgataagga tcttcctaga gcatggctac gtagataagt agcatggcgg
     6901 gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     6961 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     7021 ggcctcagtg agcgagcgag cgcgcagctg cattaatgaa tcggccaacg cgcggggaga
     7081 ggcggtttgc gtattgggcg ct
//
