LOCUS       sequence-274426-        7567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  VAChT tagged with SNAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7567)
  AUTHORS   Martineau M, Somasundaram A, Grimm JB, Gruber TD, Choquet D, Taraska
            JW, Lavis LD, Perrais D
  TITLE     Semisynthetic fluorescent pH sensors for imaging exocytosis and 
            endocytosis.
  JOURNAL   Nat Commun. 2017 Nov 10;8(1):1412. doi: 10.1038/s41467-017-01752-5.
  PUBMED    29123102
REFERENCE   2  (bases 1 to 7567)
  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: "Nat 
            Commun."; date: "2017-11-10"; volume: "8(1):1412. doi"; pages: " 
            10.1038/s41467-017-01752-5"
COMMENT     Sequence Label: VAChT-SNAP
FEATURES             Location/Qualifiers
     source          1..7567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      964..973
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1198..1743
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for mammalian expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(3158..3175)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3164..3388
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3434..3862
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3521..3540)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3731..3752
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3871..3891)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3876..4205
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4056..4191
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4118..4137
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4272..5066
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4326..4345)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4936..4955
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5240..5361
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5277..5296)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5331..5350
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5410..5426)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5410..5426)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5423..5445)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5434..5450
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5458..5464,5465..5482,5483..5488))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5503..5524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5641..5658)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5812..6400)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5892..5911)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6571..7362,7363..7431))
                     /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     7194..7213
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7432..7536)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcacg
      961 cgtgccacca tggaacccac cgcgccaacc ggtcaggccc gggcggcggc caccaaactg
     1021 tcggaagcgg tgggagccgc gctacaagag ccccagaggc agcggcgcct ggtgctggtc
     1081 atcgtgtgcg ttgcactgtt actggacaac atgttgtaca tggtcatcgt gcccattgtt
     1141 cccgactata tcgcccacat gcgcgggggc agcgagggcc cgaccctggt cacgcgtatg
     1201 gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa gctggaactg
     1261 tctgggtgcg aacagggcct gcacgagatc aagctgctgg gcaaaggaac atctgccgcc
     1321 gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc actgatgcag
     1381 gccaccgcct ggctcaacgc ctactttcac cagcctgagg ccatcgagga gttccctgtg
     1441 ccagccctgc accacccagt gttccagcag gagagcttta cccgccaggt gctgtggaaa
     1501 ctgctgaaag tggtgaagtt cggagaggtc atcagctacc agcagctggc cgccctggcc
     1561 ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc cgtgcccatt
     1621 ctgatcccct gccaccgggt ggtgtctagc tctggcgccg tggggggcta cgagggcggg
     1681 ctcgccgtga aagagtggct gctggcccac gagggccaca gactgggcaa gcctgggctg
     1741 ggtccgaccc tggtctctga ggtgtgggaa cccactctgc cgccgcccac tctggctaat
     1801 gccagtgcct acttggccaa cacgtcggcg tccccgacgg ctgccgggtc ggctcggtca
     1861 atcctgcgac ctcgctaccc cacagaaagc gaagatgtga agataggtgt gctgtttgcc
     1921 tccaaggcta tcctgcagct tctggtgaac cccttaagcg ggcctttcat tgatcgcatg
     1981 agctacgacg tgccgctgct tataggcctg ggcgtcatgt tcgcctccac agtcatgttt
     2041 gcctttgcag aagactatgc cacgctcttc gctgcgcgca gtctacaagg cctgggctcg
     2101 gccttcgcgg acacgtctgg cattgccatg atcgccgaca agtatcccga ggagcctgag
     2161 cgcagtcgtg ccctgggcgt ggcgctagcc tttattagct ttggaagcct agtggcgcca
     2221 ccgtttgggg gcatcctcta cgagttcgcg ggcaagcgtg taccctttct agtgctcgcc
     2281 gctgtgtccc ttttcgacgc gctcctgctc ctggcggtgg ctaagccctt ctcggctgcg
     2341 gctcgggcgc gagccaacct gccggtgggc acacctatcc atcgcctcat gctagaccct
     2401 tacatcgctg tggtagccgg cgcgctcacc acttgtaaca ttccccttgc gttcctcgag
     2461 cccaccatag ccacgtggat gaagcacaca atggccgcat ccgagtggga gatgggcatg
     2521 gtttggctgc cggctttcgt gccacacgtg ttaggcgtct acctcaccgt gcgcctggcg
     2581 gcgcgttatc cacacctgca gtggctgtac ggcgctctcg ggctagcggt aattggagtg
     2641 agctcttgcg tcgtacctgc ctgtcgctca ttcgcgccgt tagtggtctc gctctgcgga
     2701 ctctgcttcg gcatcgcgtt agtggacaca gcgctcctac ccacgctcgc ctttctggtg
     2761 gacgtgcgcc acgtatccgt ctatggcagt gtctatgcca tagctgacat ctcctattct
     2821 gtggcctacg cgctcgggcc catagtggca ggccacatcg ttcactctct tggctttgag
     2881 cagctcagcc tgggcatggg cctggccaac ctgctctacg caccagtcct tcttcttttg
     2941 cgcaatgtag gcctccttac acgctcgcgt tcggagcgcg atgtgttgct tgatgaaccg
     3001 ccgcagggtc tgtacgacgc ggtgcgcctg cgtgaggtgc agggcaagga tggcggcgaa
     3061 ccttgtagcc cacctggccc ttttgacggg tgcgaggacg actacaacta ttactcccgc
     3121 agctagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3181 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3241 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3301 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3361 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3421 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3481 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3541 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3601 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3661 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3721 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3781 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3841 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3901 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3961 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4021 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     4081 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4141 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4201 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4261 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4321 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4381 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4441 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4501 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4561 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4621 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4681 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4741 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4801 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4861 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4921 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4981 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5041 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     5101 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5161 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5221 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5281 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5341 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5401 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5461 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5521 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5581 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5641 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5701 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5761 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5821 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5881 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5941 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6001 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6061 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6121 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6181 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6241 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6301 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6361 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6421 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6481 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6541 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6601 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6661 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6721 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6781 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6841 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6901 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6961 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     7021 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     7081 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     7141 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7201 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7261 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7321 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7381 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7441 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7501 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7561 tgacgtc
//
