LOCUS       sequence-435246-        4897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a GST fusion to vGlut peptide for immunoisolation of 
            glutamatergic vesicles.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4897)
  AUTHORS   Leitz J, Wang C, Esquivies L, Peters JJ, Gopal N, Pfuetzner RA, Wang
            AL, Brunger AT
  TITLE     Observing isolated synaptic vesicle association and fusion ex vivo.
  JOURNAL   Nat Protoc. 2024 Jul 2. doi: 10.1038/s41596-024-01014-x.
  PUBMED    38956381
REFERENCE   2  (bases 1 to 4897)
  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 Protoc.
            2024 Jul 2. doi: 10.1038/s41596-024-01014-x."
COMMENT     Sequence Label: vGlut1 peptide- GST
FEATURES             Location/Qualifiers
     source          1..4897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      27..615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     516..535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     769..786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     884..901
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     885..901
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1364..1383
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1364..1382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1383..1407
                     /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)."
     CDS             1462..2115
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2073..2095
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2122..2145
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2149..2172
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     terminator      2365..2412
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2635..2654)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2660..2679)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2721..2812
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2813..2881,2882..3673)
                     /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(3031..3050)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3756..4838
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3775..3794)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
       61 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
      121 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
      181 gtgtagccgt agttagccca ccacttcaag aactctgtag caccgcctac atacctcgct
      241 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      301 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      361 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
      421 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      481 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      541 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      601 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      661 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      721 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      781 agcgaggaag cggaaggcga gagtagggaa ctgccaggca tcaaactaag cagaaggccc
      841 ctgacggatg gcctttttgc gtttctacaa actctttctg tgttgtaaaa cgacggccag
      901 tcttaagctc gggccccctg ggcggttctg ataacgagta atcgttaatc cgcaaataac
      961 gtaaaaaccc gcttcggcgg gtttttttat ggggggagtt tagggaaaga gcatttgtca
     1021 gaatatttaa gggcgcctgt cactttgctt gatatatgag aattatttaa ccttataaat
     1081 gagaaaaaag caacgcactt taaataagat acgttgcttt ttcgattgat gaacacctat
     1141 aattaaacta ttcatctatt atttatgatt ttttgtatat acaatatttc tagtttgtta
     1201 aagagaatta agaaaataaa tctcgaaaat aataaaggga aaatcagttt ttgatatcaa
     1261 aattatacat gtcaacgata atacaaaata taatacaaac tataagatgt tatcagtatt
     1321 tattatcatt tagaataaat tttgtgtcgc ccttccgcga aattaatacg actcactata
     1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttttgaga
     1441 ccttaaagaa ggagatataa aatgtcccca atcctcggct actggaagat caaaggcctc
     1501 gtgcagccca ctcgacttct tttggaatat cttgaagaaa aatatgagga acatttgtat
     1561 gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc
     1621 aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt
     1681 tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca
     1741 atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa
     1801 gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc
     1861 gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc
     1921 atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc
     1981 ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg
     2041 aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc
     2101 gaccatcctc caaaatcgga tctggaagtt ctttttcagg gtcctatgtt agaggttctg
     2161 tttcaaggtc caccaggtat ttcgggcggc ggcggcggta ttttgggttc cgacgagagc
     2221 gagatggaag atgaagcgga gccgccgggt gcgccgccgg caccgccgcc gagctatggt
     2281 gccacccaca gcacggtgca gccgccgcgt ccgcctccgc cggttcgcga ctactgataa
     2341 ggttagataa tagggtctca ccccctagca taaccccttg gggcctctaa acgggtcttg
     2401 aggggttttt tgcccctgag acgcgtcaat cgagttcgta cctaagggcg acaccccata
     2461 attagcccgg gcgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg
     2521 cagttcccta ctctcgcatg gggagtcccc acactaccat cggcgctacg gcgtttcact
     2581 tctgagttcg gcatggggtc aggtgggacc accgcgctac tgccgccagg caaacaaggg
     2641 gtgttatgag ccatattcag gtataaatgg gctcgcgata atgttcagaa ttggttaatt
     2701 ggttgtaaca ctgaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2761 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga atatgagtat
     2821 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2881 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2941 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3001 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3061 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3121 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3181 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3241 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3301 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3361 gatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3421 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3481 tccggctggc tggtttattg ctgataaatc cggagccggt gagcgtggtt ctcgcggtat
     3541 catcgcagcg ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3601 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3661 taagcattgg taagcggcgc gccatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     3721 agcgcccgga agagagtcaa ttcagggtgg tgaatatgaa accagtaacg ttatacgatg
     3781 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     3841 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     3901 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     3961 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     4021 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     4081 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     4141 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     4201 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaggac ggtacgcgac
     4261 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     4321 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     4381 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     4441 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     4501 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     4561 cggtagtggg atacgacgat accgaagata gctcatgtta tatcccgccg ttaaccacca
     4621 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     4681 agggccaggc ggtgaagggc aatcagctgt tgccagtctc actggtgaaa agaaaaacca
     4741 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     4801 tggcacgaca ggtttcccga ctggaaagcg ggcagtgact catgaccaaa atcccttaac
     4861 gtgagttacg cgcgcgtcgt tccactgagc gtcagac
//
