LOCUS       sequence-421660-        6908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SUMO-tagged rat FL-syt1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6908)
  AUTHORS   Courtney KC, Mandal T, Mehta N, Wu L, Li Y, Das D, Cui Q, Chapman ER
  TITLE     Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation
            of large, stable fusion pores via robust membrane penetration.
  JOURNAL   Nat Commun. 2023 Nov 27;14(1):7761. doi: 10.1038/s41467-023-42497-8.
  PUBMED    38012142
REFERENCE   2  (bases 1 to 6908)
  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: "2023-11-27"; volume: "14(1):7761. doi"; pages: " 
            10.1038/s41467-023-42497-8"
COMMENT     Sequence Label: pET SUMO-FL-syt1
FEATURES             Location/Qualifiers
     source          1..6908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      5..593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     494..513
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     747..764
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    779..918
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(844..863)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1004..1026
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1020..1211)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2020..3102)
                     /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     3064..3083
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3103..3180)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3386..3405
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3489..3508
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3489..3507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3508..3532
                     /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)."
     RBS             3547..3569
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3589..3606
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3616..3633
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3640..3933
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             5261..5278
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5331..5349)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5345..5392
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5429..5884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5516..5535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5726..5747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5976..6791)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5976..6791)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6699..6718
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6783..6802
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
       61 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
      121 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
      181 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
      241 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
      301 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
      361 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
      421 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      481 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      541 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      601 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
      661 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
      721 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
      781 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
      841 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
      901 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
      961 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     1021 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     1081 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     1141 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     1201 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     1261 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     1321 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     1381 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     1441 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     1501 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     1561 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     1621 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     1681 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     1741 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     1801 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     1861 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     1921 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     1981 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2041 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2101 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     2161 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     2221 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     2281 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     2341 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     2401 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     2461 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     2521 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     2581 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     2641 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     2701 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     2761 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     2821 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     2881 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     2941 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     3001 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     3061 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     3121 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     3181 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     3241 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     3301 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     3361 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     3421 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     3481 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     3541 aataattttg tttaacttta agaaggagat atacatatgg gcagcagcca tcatcatcat
     3601 catcacggca gcggcctggt gccgcgcggc agcgctagca tgtcggactc agaagtcaat
     3661 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag
     3721 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg
     3781 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac
     3841 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat
     3901 attattgagg ctcacagaga acagattggt ggatccatgg tgagtgccag tcatcctgag
     3961 gccctggccg cccctgtcac cactgttgcg acccttgtcc cacacaatgc cactgagcca
     4021 gccagtcctg gggaagggaa ggaagatgcc ttttccaagc tgaagcagaa gtttatgaat
     4081 gagctgcata aaattccatt gccaccgtgg gccttaatag ccatagccat agttgcggtc
     4141 cttttagtcg taacctgctg cttttgtgtc tgtaagaaat gtttgttcaa aaagaaaaac
     4201 aagaagaagg ggaaggaaaa gggaggaaag aacgccatta acatgaaaga cgtgaaagac
     4261 ttagggaaga ccatgaagga tcaggccctt aaggatgacg atgctgaaac cggactgact
     4321 gatggagaag aaaaggaaga gcccaaggaa gaggagaaac tgggaaagct ccaatattca
     4381 ctggactatg acttccagaa taaccagctg ttggtgggaa tcatccaggc tgctgaactg
     4441 cccgccctgg acatgggggg tacatccgat ccatacgtca aagtcttcct gctgcctgac
     4501 aaaaagaaga aatttgagac taaagtccac cggaaaaccc tcaatccagt cttcaatgaa
     4561 caatttactt tcaaggtacc ctactcggaa ttaggtggca aaaccctggt gatggctgtg
     4621 tatgactttg atcgcttctc caagcacgac atcatcggag agttcaaagt tcctatgaac
     4681 accgtggatt ttggccatgt gaccgaggag tggcgcgatc tccagagcgc tgagaaagaa
     4741 gagcaagaga aactgggtga catctgcttc tccctccgct acgtccctac tgccggcaaa
     4801 ctgactgttg tcattctgga agccaagaac ctgaagaaga tggatgtggg tggcttatct
     4861 gatccctacg tgaagattca cctgatgcag aacggtaaga ggctgaagaa gaaaaagacg
     4921 acgattaaga agaacacact caacccctac tacaacgagt ccttcagctt tgaagttccg
     4981 ttcgagcaaa tccagaaagt gcaagtggtg gtaactgttt tggactatga caagattggc
     5041 aagaacgacg ccatcggaaa agtcttcgtt ggttacaaca gcactggggc ggagctgcga
     5101 cactggtcag acatgctggc caacccccgg cgacccatcg cacagtggca cactctgcag
     5161 gtagaggagg aggttgatgc catgctggct gtcaagaagt aaaagcttag gtatttattc
     5221 ggcgcaaagt gcgtcgggtg atgctgccaa cttagtcgag caccaccacc accaccactg
     5281 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     5341 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     5401 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     5461 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5521 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5581 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5641 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5701 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5761 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5821 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5881 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5941 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     6001 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     6061 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     6121 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     6181 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     6241 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     6301 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     6361 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     6421 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     6481 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     6541 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     6601 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     6661 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     6721 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     6781 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     6841 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6901 caaaggat
//
