LOCUS       sequence-385123-        6066 bp    DNA     circular SYN 14-APR-2025
DEFINITION  AAV plasmid for mammalian Cre-dependent expression of 
            PKI-alpha-mut-P2A-mRuby3 under a CAG promotor w/ WPRE enhancer. 
            PKI-alpha-mut bears the R20G/R21G mutation that renders it 
            non-functional..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6066)
  AUTHORS   Ma L, Day-Cooney J, Benavides OJ, Muniak MA, Qin M, Ding JB, Mao T, 
            Zhong H
  TITLE     Locomotion activates PKA through dopamine and adenosine in striatal 
            neurons.
  JOURNAL   Nature. 2022 Nov;611(7937):762-768. doi: 10.1038/s41586-022-05407-4.
            Epub 2022 Nov 9.
  PUBMED    36352228
REFERENCE   2  (bases 1 to 6066)
  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: 
            "10.1038/s41586-022-05407-4"; journalName: "Nature"; date: 
            "2022-11"; volume: "611"; issue: "7937"; pages: "762-768"
COMMENT     Sequence Label: pAAV_CAG_FLEX_PKI-alpha-mut-P2A-mRuby3_WPRE
FEATURES             Location/Qualifiers
     source          1..6066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   35..164
                     /label=AAV2 ITR
     promoter        475..752
                     /label=chicken beta-actin promoter
     primer_bind     1031..1053
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1115..1134
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1169..1202
                     /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    1265..1298
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1314..2024)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     regulatory      2021..2030
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2037..2093)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      2330..2339
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2365..2398)
                     /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(2469..2502)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2544..3132
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2597..2617)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3015..3026)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3175..3296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3186..3205)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3240..3259
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3458..3587
                     /label=AAV2 ITR
     rep_origin      complement(3824..4412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3904..3923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4583..5374,5375..5443))
                     /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     5206..5225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5444..5548)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5574..6029)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5711..5732)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5923..5942
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcgatcggtg cgggcctctt cgctattacg ccagctgcgc gctcgctcgc tcactgaggc
       61 cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
      121 agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt taatgattaa
      181 cccgccatgc tacttatcta cgtagccatg ctctaggaag agtaccattg acgtcaataa
      241 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      301 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      361 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      421 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      481 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      541 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
      601 cgccrggsgg ggsggggsgg ggsgrggggs ggggsggggc grggcggaga ggtgcggcgg
      661 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      721 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
      781 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
      841 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat
      901 gacggcttgt ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt
      961 tgtgcggggg gagcggctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg
     1021 ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc tgctaaccat
     1081 gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct
     1141 catcattttg gcaaagaatt ggatccccat aacttcgtat aaagtatcct atacgaagtt
     1201 atatcaaaat aggaagacca atgcttcacc atcgacccga attgccaagc atcaccatcg
     1261 acccataact tcgtataatg tatgctatac gaagttatac taggatcccc ttacttgtac
     1321 agctcgtcca tgccaccacc aaggttgctg tatttggcaa ctgccacttc tctttgcact
     1381 acgtaggttt cattgtcact ctcctcgatc ctttccaggc ggtgatcaac ggcatggaca
     1441 ccgggcatct tgatgttccc gacggtcttt tttgacctgt aagttgtcac gaagttgcag
     1501 tgcaggtggc caccaccatc aactttcagt gcgatgtcag tgtatcctct cagaccacca
     1561 tctgctggat acatcatctc tgtattaggc tcccaaccct tggtcttctt ctgcatcacg
     1621 ggaccattgg agggaaagtt tacccctctg accttgacgt tgtagacgag ctcgccatcc
     1681 tcaaggctgg tgtcctgcgt gacggtgacg actccaccat cttcgtatct cgtaactctt
     1741 tcccaagtaa aaccctcagg aaaggactgt ttaaagaaat cagggatgtc ggccgggtac
     1801 ttgataaagg tacggctgcc atacatgaac gacgtggcaa gaatgtcaaa ggcaaatggc
     1861 aggggtcctc cctcgatgac tttgatcctc atggtttgca ctccctcgta cggtctgcct
     1921 tctccttcac ctgtgcattt gaattggtgg ccgttgaccg aaccttccat gaccaccttc
     1981 atacgcatat tttccttgat cagctcttcg cccttagaca ccatggtggc ggatccaggg
     2041 ccagggttct cttccacgtc gcctgcctgc ttcagcaggc tgaagttggt ggctccagag
     2101 ccgctttcag acttggctgc ttctccctgg gcttctccac tttgttcggt ggagcttctc
     2161 tgtccatcat cttcaccttc tgtcttgttg atatcaaggc ctgctagttt taaggctaat
     2221 tcattgctgt tgccacttgc agaggaaacc aggatatcat gtattgcatt tcctccacct
     2281 gttcttcctg aagcaatgaa atctgcatac gtagtttcca catcagttcc catggtggcg
     2341 gcactagtgg taccgaattc tagtataact tcgtatagga tactttatac gaagttatca
     2401 ttgggattct tcctattttg atccaagcat caccatcgac cctctagtcc agatctcacc
     2461 atcgacccat aacttcgtat agcatacatt atacgaagtt atgtccctcg aagaggttcg
     2521 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     2581 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     2641 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     2701 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     2761 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     2821 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     2881 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     2941 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     3001 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     3061 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     3121 ccgcctcccc gcatcgatac cgtcgacccg ggcggccgct tcgagcagac atgataagat
     3181 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     3241 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     3301 acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa
     3361 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tcttcctaga gcatggctac
     3421 gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg
     3481 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3541 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cattaatgaa
     3601 tcggccaacg cgcggggaga ggcggtttgc gtattkggcg ctcttccgct tcctcgctca
     3661 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3721 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3781 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3841 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3901 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3961 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4021 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4081 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4141 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4201 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4261 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4321 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4381 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4441 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4501 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4561 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4621 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4681 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4741 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4801 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4861 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4921 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4981 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5041 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5101 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5161 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5221 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5281 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5341 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5401 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5461 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5521 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5581 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5641 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5701 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5761 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5821 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5881 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5941 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     6001 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     6061 ggaagg
//
