LOCUS       sequence-414435-        6828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses wild-type Octopus synaptotagmin-1 C2A domain in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6828)
  AUTHORS   Birk MA, Liscovitch-Brauer N, Dominguez MJ, McNeme S, Yue Y, Hoff 
            JD, Twersky I, Verhey KJ, Sutton RB, Eisenberg E, Rosenthal JJC
  TITLE     Temperature-dependent RNA editing in octopus extensively recodes the
            neural proteome.
  JOURNAL   Cell. 2023 Jun 8;186(12):2544-2555.e13. doi: 
            10.1016/j.cell.2023.05.004. Epub 2023 Jun 8.
  PUBMED    37295402
REFERENCE   2  (bases 1 to 6828)
  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: 
            "10.1016/j.cell.2023.05.004"; journalName: "Cell"; date: "2023-06-8-
            8"; volume: "186"; issue: "12"; pages: "2544-2555.e13"
COMMENT     Sequence Label: p202-Syt1-C2A-wt
FEATURES             Location/Qualifiers
     source          1..6828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    39..63
                     /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             78..100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             113..130
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             131..1225
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="IEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK
                     FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIA
                     YPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAAD
                     GGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETA
                     MTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLEN
                     YLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWY
                     AVRTVVINAASGRQTVDEALKDAQT"
     primer_bind     1199..1222
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1262..1273
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1277..1297
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1712..1729
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1782..1800)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1796..1843
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1880..2335
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1967..1986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2177..2198
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2427..3242)
                     /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(2427..3242)
                     /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     3150..3169
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3234..3253
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3364..3952
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3853..3872
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4106..4123
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4138..4277
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4203..4222)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4363..4385
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4379..4570)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5379..6461)
                     /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     6423..6442
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6462..6539)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6745..6764
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
       61 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg atcatcatca
      121 tcatcatcat atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      181 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      241 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      301 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      361 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      421 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
      481 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
      541 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
      601 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
      661 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
      721 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
      781 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
      841 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
      901 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcaaaaga
      961 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1021 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     1081 cgccacaatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1141 tttctggtat gccgtgcgta ctgtggtgat caacgccgcc agcggtcgtc agactgtcga
     1201 tgaagccctg aaagacgcgc agactaattc gagctcagat tatgatattc cgaccaccgg
     1261 gatcgaggga aggggagaaa atctttattt tcaaggtgat attactcata tggtgaagtt
     1321 ggggaaattg caatactcta tggattacga tttccaaaaa ggagaactta ccgtaaatgt
     1381 catccaggca gccgatttac cgggcatgga tatgtctgga acttccgacc cgtatgtcaa
     1441 agtttatctg atgcccgaca aaaagaaaaa atttgaaacg aaagtacacc gtaaaacgtt
     1501 gaatccagtg tttaacgaga gctttacttt caagaatgtg ccttatgccg acattacggg
     1561 aaaaacgctg atcttcgcta tttacgactt tgaccgtttt tccaagcacg atcaaattgg
     1621 ccaagtccag gttccgatga attccatcga tcttggttct gtcatcgaag aatggcgcga
     1681 tttaacgtca ccagacaact agtaactcga gcaccaccac caccaccact gagatccggc
     1741 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1801 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1861 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1921 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1981 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2041 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2101 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2161 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2221 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2281 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     2341 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2401 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     2461 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     2521 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     2581 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     2641 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     2701 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     2761 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     2821 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2881 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     2941 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     3001 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     3061 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     3121 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     3181 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     3241 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     3301 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3361 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3421 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3481 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3541 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3601 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3661 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3721 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3781 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3841 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3901 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3961 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4021 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4081 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     4141 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     4201 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     4261 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4321 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4381 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     4441 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     4501 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     4561 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     4621 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     4681 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4741 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4801 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4861 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4921 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4981 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     5041 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     5101 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     5161 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     5221 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     5281 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     5341 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5401 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5461 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     5521 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5581 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     5641 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     5701 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5761 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5821 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5881 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5941 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     6001 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     6061 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     6121 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6181 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     6241 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     6301 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     6361 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     6421 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     6481 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     6541 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     6601 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     6661 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6721 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6781 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcga
//
