LOCUS       sequence-412306-        6675 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Monitoring neuropeptide release.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6675)
  AUTHORS   Kim D-I, Park S, Ye M, Chen JY, Jhang J, Hunker AC, Zweifel LS, 
            Palmiter RD, Han S
  TITLE     Novel genetically encoded tools for imaging or silencing 
            neuropeptide release from presynaptic terminals in vivo
  JOURNAL   bioRxiv 2023.01.19.524797
REFERENCE   2  (bases 1 to 6675)
  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: "bioRxiv 
            2023.01.19.524797"
COMMENT     Sequence Label: pAAV-hSyn1-FLEX-CybSEP2-WPRE
FEATURES             Location/Qualifiers
     source          1..6675
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        166..613
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(646..679)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(690..723)
                     /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)."
     CDS             complement(735..758)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1170..1886)
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             complement(1911..2627)
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     regulatory      2963..2972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2978..3011
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3022..3055
                     /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)."
     misc_feature    3079..3667
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3132..3152)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3550..3561)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3702..3719)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3708..3915
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3938..4078
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3938..4067
                     /label=AAV2 ITR
     rep_origin      4153..4608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4240..4259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4450..4471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4625..4644)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4744..4766
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4804..4822)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4890..4994
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4995..5063,5064..5855)
                     /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(5213..5232)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6026..6614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6515..6534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtctgcag agggccctgc gtatgagtgc aagtgggttt
      181 taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga
      241 caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa
      301 caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc
      361 cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc
      421 cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc
      481 agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg
      541 cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg
      601 cctgagagcg cagacgcgtg gacggtaccc ttcaggtcga cgtcgataac ttcgtatagc
      661 atacattata cgaagttatc cgaatcgcaa taacttcgta taggatactt tatacgaagt
      721 tatttggcgc gtcacttatc gtcgtcatcc ttgtaatcct ggggactggg gctgtctccc
      781 tccgtgagtg tcttgaagtc catggagagg gcttgctctt cagcctggga aggccgcttc
      841 cagtcagcct gagccaagat gtagagcaca accaccccaa aacagaccag cagcaagcct
      901 agcacgttgg ccagcacccc ctcaggttca aacgtgctgt acttggaccc caatttaaac
      961 agcagggcct ccttcaggcc cagtagggct gtgcccacag agaagaggaa gatggtggca
     1021 ccaaagaaaa tggcctgagg gcggtagcgg ctccgcagag agaatgaagc tccagggaac
     1081 aggaagaagc tgaaacccac gagccactgc acaaagtaca gaacaaagac taagatccca
     1141 caccagctgt gcaagctgta caggtctgct ttgtatagtt catccatgcc atgtgtaatc
     1201 ccagcagctg ttacaaactc aagaaggacc atgtggtctc tcttttcgtt gggatctttc
     1261 gaaagagtag aagttgtaaa caggtaatgg ttgtctggta aaaggacggg cccatcgcca
     1321 attggagtat tttgttgata atggtctgct agttgaacgc ctccatcttc aatgttgtgt
     1381 ctaattttga agttagcttt gattccattc ttttgtttgt ctgccatgat gtacacctgg
     1441 tgatcgttat agttgtattc caatttgtgt ccaagaatgt ttccatcttc tttaaaatca
     1501 atacctttta actcgattct attaacaagg gtatcacctt caaacttgac ttcagcacgt
     1561 gtcttgtagt tcccgtcatc tttgaaaaat atagttcttt cctgtacata accttcgggc
     1621 atggcactct tgaaaaagtc atgccgtttc atatgatctg ggtatcttga aaagcattga
     1681 acaccataag ttaaagtagt gacaagtgtt ggccatggaa caggtagttt tccagtagtg
     1741 caaataaatt taagggtaag ttttccgtat gttgcatcac cttcaccctc tccactgaca
     1801 gaaaatttgt gcccattaac atcaccatct aattcaacaa gaattgggac aactccagtg
     1861 aaaagttctt ctcctttact tcccatatag cccttcttct tgtggtagtc tttgtatagt
     1921 tcatccatgc catgtgtaat cccagcagct gttacaaact caagaaggac catgtggtct
     1981 ctcttttcgt tgggatcttt cgaaagagta gaagttgtaa acaggtaatg gttgtctggt
     2041 aaaaggacgg gcccatcgcc aattggagta ttttgttgat aatggtctgc tagttgaacg
     2101 cctccatctt caatgttgtg tctaattttg aagttagctt tgattccatt cttttgtttg
     2161 tctgccatga tgtacacctg gtgatcgtta tagttgtatt ccaatttgtg tccaagaatg
     2221 tttccatctt ctttaaaatc aatacctttt aactcgattc tattaacaag ggtatcacct
     2281 tcaaacttga cttcagcacg tgtcttgtag ttcccgtcat ctttgaaaaa tatagttctt
     2341 tcctgtacat aaccttcggg catggcactc ttgaaaaagt catgccgttt catatgatct
     2401 gggtatcttg aaaagcattg aacaccataa gttaaagtag tgacaagtgt tggccatgga
     2461 acaggtagtt ttccagtagt gcaaataaat ttaagggtaa gttttccgta tgttgcatca
     2521 ccttcaccct ctccactgac agaaaatttg tgcccattaa catcaccatc taattcaaca
     2581 agaattggga caactccagt gaaaagttct tctcctttac ttcccatata gcccttcttc
     2641 ttgtggtagt cgaacacagc caccaacccc accagggcga tgatgaaggc gaagacgtgc
     2701 agcagcccag ccaagatctt ggtcgtgcgt ttggcttctc ttctgaagac acggtacacc
     2761 agcagagcat ctccttgaag gaagatcatg cctatgacca tgcagagcgg gtgcacatta
     2821 aactgcaggg agctttccca ggcaatgcca cctcggtaca gacccagcca cgcaccagtc
     2881 acagccacta cagtgaggcc cagcagctgg gagaaggcca catagtacgg cagtgcagca
     2941 gggacagacg cagaactgtg ctccatggtg gcccttgata acttcgtata atgtatgcta
     3001 tacgaagtta tccgaatcgc aataacttcg tataaagtat cctatacgaa gttatcgaag
     3061 atcatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3121 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3181 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3241 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3301 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3361 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3421 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3481 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     3541 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3601 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3661 tccccgcatc gataccgagc gctgctcgag agatcgatct gcctcgactg tgccttctag
     3721 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3781 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3841 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3901 caggcatgct ggggacacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     3961 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4021 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4081 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4141 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4201 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     4261 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4321 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4381 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4441 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     4501 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     4561 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4621 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4681 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4741 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4801 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4861 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4921 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4981 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5041 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5101 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5161 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5221 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5281 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5341 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5401 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5461 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5521 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5581 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5641 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5701 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5761 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5821 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5881 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5941 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6001 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6061 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6121 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6181 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6241 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6301 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6361 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6421 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6481 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6541 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6601 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6661 cttttgctca catgt
//
