LOCUS       sequence-229642-        4606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for structure determination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4606)
  AUTHORS   Iwai H, Mikula KM, Oeemig JS, Zhou D, Li M, Wlodawer A
  TITLE     Structural Basis for the Persistence of Homing Endonucleases in 
            Transcription Factor IIB Inteins.
  JOURNAL   J Mol Biol. 2017 Dec 8;429(24):3942-3956. doi: 
            10.1016/j.jmb.2017.10.016. Epub 2017 Oct 18.
  PUBMED    29055778
REFERENCE   2  (bases 1 to 4606)
  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.jmb.2017.10.016"; journalName: "J Mol Biol"; date: 
            "2017-12-8- 8"; volume: "429"; issue: "24"; pages: "3942-3956"
COMMENT     Sequence Label: pSCFRSF131
FEATURES             Location/Qualifiers
     source          1..4606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             124..414
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIETHREQIGG"
     CDS             976..1131
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             1160..1204
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1242..1260)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1256..1303
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1533..2348)
                     /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(1533..2348)
                     /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     2256..2275
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2349..2440)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2456..3205)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(2466..2483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2706..2725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3401..4483)
                     /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     4445..4464
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4484..4561)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 actagtgact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
      181 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag aaatcttctt taagatcaaa
      241 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
      301 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat
      361 ttggacatgg aggataacga tattattgag actcacagag aacagattgg tggatccggt
      421 ggatatgctg ttgattacaa cgaaccaata atcattaaag agaatggaga aataaaagtt
      481 gttaaaattg gagaacttat agataaaatt attgaaaact cagagaatat tagaagagag
      541 ggcatcttag agatagcaaa atgtaaaggt attgaagtta ttgcctttaa cagcaattac
      601 aaatttaaat tcatgcctgt ttcggaggtt tcaaggcatc cagttagtga gatgtttgaa
      661 atagttgttg aagggaataa aaaggttaga gttaccagaa gccatagtgt ctttaccata
      721 agagataatg aggtagttcc aataagagtt gatgagctaa aagttggaga tatattagtt
      781 ttagcaaaaa gaataacaaa tatatatacc aatagaaaac tcgaaaaact tataaactct
      841 gatttcatat tcttaaaaat taaagagatt aataaggtag agccaaccag tggatatgcc
      901 tatgatttaa ctgttccaaa tgcagaaaac ttcgttgctg gatttggagg atttgtatta
      961 cacaacgcct aaggtaccac aaatcctggt gtatccgctt ggcaggtcaa cacagcttat
     1021 actgcgggac aattggtcac atataacggc aagacgtata aatgtttgca gccccacacc
     1081 tccttggcag gatgggaacc atccaacgtt cctgccttgt ggcagcttca ataagcttgc
     1141 ggccgcactc gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga
     1201 ctcgtctact agcgcagctt aattaaccta ggctgctgcc accgctgagc aataactagc
     1261 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt
     1321 tgagaagcac acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac
     1381 ataagcggct atttaacgac cctgccctga accgacgaca agctgacgac cgggtctccg
     1441 caagtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1501 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     1561 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     1621 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     1681 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     1741 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     1801 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     1861 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg
     1921 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     1981 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     2041 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     2101 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     2161 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     2221 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     2281 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     2341 tggctcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     2401 agcggataca tatttgaatg tatttagaaa aataaacaaa taggcatgca gcgctcttcc
     2461 gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggtgtcagct
     2521 cactcaaaag cggtaatacg gttatccaca gaatcagggg ataaagccgg aaagaacatg
     2581 tgagcaaaaa gcaaagcacc ggaagaagcc aacgccgcag gcgtttttcc ataggctccg
     2641 cccccctgac gagcatcaca aaaatcgacg ctcaagccag aggtggcgaa acccgacagg
     2701 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2761 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2821 tagctcacgc tgttggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2881 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2941 caacccggta agacacgact tatcgccact ggcagcagcc attggtaact gatttagagg
     3001 actttgtctt gaagttatgc acctgttaag gctaaactga aagaacagat tttggtgagt
     3061 gcggtcctcc aacccactta ccttggttca aagagttggt agctcagcga accttgagaa
     3121 aaccaccgtt ggtagcggtg gtttttcttt atttatgaga tgatgaatca atcggtctat
     3181 caagtcaacg aacagctatt ccgttactct agatttcagt gcaatttatc tcttcaaatg
     3241 tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg
     3301 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     3361 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3421 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3481 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3541 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3601 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     3661 cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc
     3721 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     3781 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     3841 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     3901 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     3961 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     4021 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     4081 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     4141 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     4201 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4261 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4321 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4381 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4441 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4501 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     4561 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaaat
//
