LOCUS       sequence-244566-        5937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of N-intein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5937)
  AUTHORS   Stevens AJ, Brown ZZ, Shah NH, Sekar G, Cowburn D, Muir TW
  TITLE     Design of a Split Intein with Exceptional Protein Splicing Activity.
  JOURNAL   J Am Chem Soc. 2016 Feb 24;138(7):2162-5. doi: 10.1021/jacs.5b13528.
            Epub 2016 Feb 8.
  PUBMED    26854538
REFERENCE   2  (bases 1 to 5937)
  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: 
            "10.1021/jacs.5b13528"; journalName: "J Am Chem Soc"; date: 
            "2016-02-24- 24"; volume: "138"; issue: "7"; pages: "2162-5"
COMMENT     Sequence Label: pET-30-His6-SUMO-AEY-CfaN
FEATURES             Location/Qualifiers
     source          1..5937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(513..806)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(813..830)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     RBS             874..896
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    911..935
                     /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)."
     primer_bind     complement(935..954)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(936..954)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1042..1061)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1267..1344
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1345..2427
                     /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(1364..1383)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3236..3427
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3421..3443)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3529..3668
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3584..3603
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3683..3700)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3854..4442)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3934..3953)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4553..4572)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4564..5379
                     /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             4564..5379
                     /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     complement(4637..4656)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5471..5926)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5608..5629)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5820..5839
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattctta cggcaaacca tcaacttgtt tcaggtctaa cccccgttcg
      241 aaaatctcat caatcggtaa catctgaccg tcggtcgtca taaatttgtg atctttcgtt
      301 gcacggataa tcgaaccgtc ttctaagcag tattcgaaaa cttcctgttc tccccggtta
      361 tgccactgag caataggttg tgtatacaca aagccatttt tatctaccgt atagactgtg
      421 cattcaatcc gttcctccac gatcttaccg ataggaagga atccatattc aacggtcaga
      481 atctctgtgt cgtaagacag gcaatattcc gcaccaccaa tctgttctct gtgagcctca
      541 ataatatcgt tatcctccat gtccaaatct tcaggggtct gatcagcttg aattctaata
      601 ccgtcgtaca agaatcttaa ggagtccatt tccttaccct gtcttttagc gaacgcttcc
      661 atcagccttc ttaaaggagt ggtctttttg atcttgaaga agatctctga agatccatcg
      721 gacaccttta aattgatgtg agtctcaggc ttgacttctg gcttgacctc tggcttagct
      781 tcttgattga cttctgagtc cgacatgcta gcgctgccgc gcggcaccag gccgctgccg
      841 tgatgatgat gatggctgct gcccatatgt atatctcctt cttaaagtta aacaaaatta
      901 tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg
      961 cgggatcgag atcgatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
     1021 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
     1081 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
     1141 ggggactgtt gggcgccatc tccttgcatg caccattcct tgtggcggcg gtgctcaacg
     1201 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag
     1261 atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     1321 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     1381 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     1441 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     1501 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     1561 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     1621 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     1681 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     1741 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     1801 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     1861 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     1921 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     1981 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     2041 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     2101 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggacatctc ggtagtggga
     2161 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     2221 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     2281 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     2341 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2401 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     2461 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     2521 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     2581 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
     2641 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
     2701 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
     2761 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     2821 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     2881 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     2941 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     3001 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     3061 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     3121 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     3181 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     3241 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     3301 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     3361 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3421 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3481 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     3541 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     3601 gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     3661 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3721 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3781 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3841 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3901 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3961 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4021 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4081 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4141 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4201 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4261 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4321 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4381 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4441 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4501 aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     4561 gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg
     4621 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     4681 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     4741 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     4801 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     4861 gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     4921 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     4981 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     5041 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     5101 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     5161 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     5221 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     5281 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     5341 aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata
     5401 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5461 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     5521 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     5581 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     5641 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     5701 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     5761 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     5821 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     5881 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
