LOCUS       sequence-412186-        7749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the green 5-HT sensor GRAB_g5-HT3.0mut in neurons in the 
            presence of Cre recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7749)
  AUTHORS   Deng F, Wan J, Li G, Dong H, Xia X, Wang Y, Li X, Zhuang C, Zheng Y,
            Liu L, Yan Y, Feng J, Zhao Y, Xie H, Li Y
  TITLE     Improved green and red GRAB sensors for monitoring spatiotemporal 
            serotonin release in vivo.
  JOURNAL   Nat Methods. 2024 Apr;21(4):692-702. doi: 
            10.1038/s41592-024-02188-8. Epub 2024 Mar 5.
  PUBMED    38443508
REFERENCE   2  (bases 1 to 7749)
  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"; doi: 
            "10.1038/s41592-024-02188-8"; journalName: "Nat Methods"; date: 
            "2024-04"; volume: "21"; issue: "4"; pages: "692-702"
COMMENT     Sequence Label: pAAV-EF1a-DIO-GRAB_g5-HT3.0mut
FEATURES             Location/Qualifiers
     source          1..7749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        101..1279
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          332..1270
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1227..1247
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1310..1343)
                     /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(1394..1427)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2033..2052
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2292..2313
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2419..2440)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3504..3528)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     sig_peptide     complement(3532..3591)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     regulatory      3588..3597
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3604..3637
                     /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    3688..3721
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3746..4334
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3799..3819)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4217..4228)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4366..4842
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4565..4584)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4882..5022
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4882..5011
                     /label=AAV2 ITR
     rep_origin      5097..5552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5184..5203)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5394..5415
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5569..5588)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5688..5710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5748..5766)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5834..5938
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5939..6007,6008..6799)
                     /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(6157..6176)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6970..7558
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7459..7478
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7620..7749
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga ggaatctttg
       61 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
      121 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      181 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
      241 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
      301 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
      361 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
      421 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
      481 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
      541 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
      601 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
      661 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
      721 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
      781 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
      841 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
      901 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
      961 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     1021 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     1081 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     1141 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     1201 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     1261 tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa taacttcgta
     1321 taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc tattagcaat
     1381 atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc gcatatgtca
     1441 ttacacactg ctaatccttt cgctaaccac actggaggga tttactggta actctaaatt
     1501 ctcaacttgc atctctatac cgggctcatt gtcactggct ttctcgatca ccggttcatt
     1561 ggtatgccga taaatgttaa cattaagctc cctcccagac aaagcagtgg cggcaactct
     1621 tggaatctgc ctgacaggag gctttttctc taccttataa ttgcaacgga ggatgatgag
     1681 gaaggcacgt ctgaaagact tattcaagaa ggcatagaga aaagggttca accccgaatt
     1741 gatatagcca agccagagga aagcagtcca cacctgctcg gggacagtgt agtctatgaa
     1801 agggtccaca atattggtga caaagaaggg ggcccagcag aaacagaagc agcccatgat
     1861 gacacataac accttggctg ccttggtctc tgtcctccag cggaagcgtg ctgctgctcc
     1921 ggtgttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt
     1981 cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta
     2041 gttgccgtcg tccttgaaga agatggtgcg ctcctgcacg tagccttcgg gcatggcgga
     2101 cttgaagaag tcgtgctgct tcatgtggtc ggggtagcga ctgaagcact gcacgccgta
     2161 ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa
     2221 cttcagggtc agcttgccat tggtggcatc gccctcaccc tcgccccgca cgctgaactt
     2281 gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc
     2341 ctcgcccttt ctcaccatgc tccctccggt accgcccttg tacagctcgt ccatgccgag
     2401 agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg
     2461 gtctttcgaa agtttggact gcacgctcag gtagtggttg tcgggcagca gcacggggcc
     2521 gtcgccgatg ggggtgttct gctggtagtg gatggcgagc tgcacgccgc cgtcctcgat
     2581 gttgtggcgg atgtggaaga tcgccttgat gccgttcttc tgcttgtcgg ccttgatata
     2641 gacgtttcct ccctcctcca ggtccagcgc gtcggtgtcg cgcggcccgg ccggcgcggg
     2701 ctcgccaggg gcgccgttga gctgggtggg cagcggttcg gcctctgcgc cccccgggcc
     2761 cgcgctgcgc tcggggccca gaccgttggg cctgcgctcg gtgccccccg gcggcgcggc
     2821 gacggcgtcc ggaccccgct tagccacgac atagattcgg taataggcca gcaccatgag
     2881 gagaaacggg atgtagaagg ccaccacaga gcaggtgata gcatagggct tgttgaccat
     2941 gaagacacac cacgtggagt tagagttgtg gctgaatttc cttttctcta tcacatcaac
     3001 tatgccgatg ttgttccagc cttgcattat ggggagaaaa gatataaaca tgggaaggac
     3061 ccagcagcct cccaacatta atgcgatgcg tagaggggtc atcttgttcc tataaaccaa
     3121 aggctggcag cagatggcgt aatacctgtc cagggaaata cagcacaggt gaaagatcga
     3181 tgctgtacaa aggtagacaa acagagaggt ccggaccagg cagaacatct ccccataagc
     3241 ccagatgtct tggaccagct caatggcacc aaagggcatc accagcaccg aaaccagcag
     3301 gtcagcaaag gcgagagaca caatgaaata gttggttttt attttcctga gctgcctgtc
     3361 cctgcacaca gccaccatca ccagcaggtt gcccaagatg gccatcagga taaccactgc
     3421 aaggaacgtg agcagcacga ccttctccac ggacctgaaa ccctcgttgg aactcacatt
     3481 agcatcaagt ttgtcaccgg tggtgccaac ttttttgtac aaacttgtgt caccagtgga
     3541 acctggaacc cagagcagca gtacccatag caggagtgtg tctgtctcca tggtggcgct
     3601 agcataactt cgtataaagt atcctatacg aagttatttg ccttaaccca gaaattatca
     3661 ctgttattct ttagaatggt gcaaagaata acttcgtata atgtatgcta tacgaagtta
     3721 tgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     3781 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3841 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3901 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3961 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     4021 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     4081 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     4141 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     4201 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     4261 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     4321 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     4381 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     4441 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     4501 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     4561 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     4621 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     4681 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     4741 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     4801 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     4861 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4921 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4981 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     5041 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     5101 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5161 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5221 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5281 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     5341 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5401 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     5461 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5521 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     5581 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     5641 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5701 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     5761 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5821 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5881 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5941 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     6001 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     6061 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     6121 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     6181 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6241 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6301 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6361 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6421 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6481 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6541 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6601 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6661 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6721 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6781 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6841 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6901 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6961 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     7021 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7081 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     7141 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7201 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7261 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7321 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7381 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7441 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7501 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7561 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc
     7621 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg
     7681 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
     7741 ggggttcct
//
