LOCUS       sequence-436595-        6521 bp    DNA     circular SYN 14-APR-2025
DEFINITION  AAV-mediated expression of improved GABA sensor (positive change in 
            fluorescence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6521)
  AUTHORS   Kolb I, Hasseman JP, Matsumoto A, Arthur BJ, Zhang Y, Tsang A, Reep 
            D, Tsegaye G, Zheng J, Patel R, Looger LL, Marvin JS, Korff WL, 
            Yonehara K, Turner GC
  TITLE     iGABASnFR2: Improved genetically encoded protein sensors of GABA
  JOURNAL   bioRxiv 2025.03.25.644953
REFERENCE   2  (bases 1 to 6521)
  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"; journalName: "bioRxiv 
            2025.03.25.644953"
COMMENT     Sequence Label: pGP-AAV-syn-flex-iGABASnFR2-WPRE
FEATURES             Location/Qualifiers
     source          1..6521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   45..174
                     /label=AAV2 ITR
     promoter        256..703
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    733..766
                     /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    829..862
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(876..1022)
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     CDS             complement(1026..1055)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     1562..1583
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1686..1707)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     sig_peptide     complement(2751..2813)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTGD"
     regulatory      2810..2819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2830..2863)
                     /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(2934..2967)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3009..3597
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3062..3082)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3480..3491)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3640..3761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3651..3670)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3705..3724
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3923..4052
                     /label=AAV2 ITR
     rep_origin      complement(4289..4877)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4369..4388)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5048..5839,5840..5908))
                     /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     5671..5690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5909..6013)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6039..6494)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6176..6197)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6388..6407
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctgcgc gctcgctcgc
       61 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag
      121 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt
      181 taatgattaa cccgccatgc tacttatcta cgtagccatg ctctaggaag atctctgcag
      241 agggccctgc gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc
      301 ctacctgacg accgaccccg acccactgga caagcaccca acccccattc cccaaattgc
      361 gcatccccta tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc
      421 agcttcagca ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct
      481 cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca
      541 ccttggtcgc gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat
      601 aggggggcac gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc
      661 tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg cagtcgaatt caagctgcta
      721 gcaaggatcc ccataacttc gtataaagta tcctatacga agttatatca aaataggaag
      781 accaatgctt caccatcgac ccgaattgcc aagcatcacc atcgacccat aacttcgtat
      841 aatgtatgct atacgaagtt atactagagc tttcaacgtg gcttcttctg ccaaagcatg
      901 atgaggatga taagggagat gatggtgagc accaccaggg ccaggatggc tgagatcacc
      961 accaccttaa agggcaagga gtgtggcacc acgatgacct cctgcgtgtc ctggcccaca
     1021 gcattcagat cctcttctga gatgagtttt tgttcgtcga cctgcagttt gactagccat
     1081 tcattccacc gtgtcgcaat ggccggaccg ttcttggccc agtaggcgaa atcgagggtg
     1141 atctgatcct tgacgtaggc ggtcggcagg ttcggtgcca ggttggcgtt ccagttgtac
     1201 tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg
     1261 cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta ggtgccgtcg
     1321 tccttgaagc tgatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag
     1381 tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg
     1441 gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc
     1501 agcttgccgt tggtggcatc gccctcgccc tcgccgcgca cgctgaactt gtggccgttt
     1561 acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg
     1621 ctcatgctcc ctccggtacc gcccttgtac agctcgtcca tgccgagagt gatcccggcg
     1681 gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagc
     1741 acggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg
     1801 gtgttctgct ggtagtggtc ggcgctctgc acgctgccgt cctccacgtt gtggcggatc
     1861 ttgaagttcg ccatgatgcc gttcttctgc ttgtcggcgg tgatccggac ctgcgcaagg
     1921 tcgaggcgct gcacactgtc gatgttcact ggagcatagg cggtcagggc ggaaaagtcg
     1981 tcctggcctt tggcgctgct ggcactggcc aggaacttca tcgcggcggc tttgttcttc
     2041 gtgcctttag gcacgacgag gatgtcggcc atcaccaggt tctgcttcca gctcacgccc
     2101 actggcgcgc cgtcttcttg cagggcgtgg atgcgaccgt tccagaactg gcccatgctg
     2161 acttcgccgg aggccagcag ctgctgcgac tgtgcaccgc cgccccacca gacgatgtct
     2221 ttcttgatgg tgtcgagttt cttgaaggcg cggtccaggt ccagcgggta gagcttgtcg
     2281 gccggtacgc cgtcggcgag cagggccagt tcgagcacgc cagggctcgg ccatttgtag
     2341 agggcgcgtt tgccggggta ggtcttggtg tcgaacagcg cggtccagtc ctggggcttg
     2401 ctggctccga gcttgccttc gttgtagccg agcacgtaag agtacaggaa ggcccccact
     2461 ccatggtcac tcacgaaccg cggatcgatc ttgtcgcgct ggatcaccga gaaatccagg
     2521 ggttcgagca ggccttcggc ggcggcgcgc aaggcgaagt cggcctctac atccaccacg
     2581 tcccactgca cgttgccgct ttcgaccatg gccttgagtt tgccgtagtc ggtgggccca
     2641 tcctggacca cggtaatgcc gctggccttg ctgaacgggt cggcccaggc ctgcttctgc
     2701 gcatcctggg tgctaccgcc ccagctcacg aaattgatgc tttcagatct gtcaccagtg
     2761 gaacctggaa cccagagcag cagtacccat agcaggagtg tgtctgtctc catggtggcg
     2821 gatcctagta taacttcgta taggatactt tatacgaagt tatcattggg attcttccta
     2881 ttttgatcca agcatcacca tcgaccctct agtccagatc tcaccatcga cccataactt
     2941 cgtatagcat acattatacg aagttatgtc cctcgaagag gttcgaattc gatatcaagc
     3001 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3061 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3121 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3181 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3241 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3301 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3361 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3421 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3481 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3541 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     3601 gataccgtcg acccgggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     3661 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3721 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3781 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct
     3841 acaaatgtgg taaaatcgat aaggatcttc ctagagcatg gctacgtaga taagtagcat
     3901 ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg
     3961 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4021 cgggcggcct cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg
     4081 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4141 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4201 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4261 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4321 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4381 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4441 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4501 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4561 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4621 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4681 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4741 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4801 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4861 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4921 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4981 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5041 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5101 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5161 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5221 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5281 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5341 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5401 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5461 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5521 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5581 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5641 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5701 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5761 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5821 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5881 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5941 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6001 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     6061 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     6121 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     6181 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6241 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     6301 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     6361 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     6421 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6481 gctacagggc gcgtcccatt cgccattcag gctgcgcaac t
//
