LOCUS       sequence-229643-        4636 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 4636)
  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 4636)
  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: pBHRSF63
FEATURES             Location/Qualifiers
     source          1..4636
                     /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             1006..1161
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             1190..1234
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1272..1290)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1286..1333
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1563..2378)
                     /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(1563..2378)
                     /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     2286..2305
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2379..2470)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2486..3235)
                     /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(2496..2513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2736..2755)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3431..4513)
                     /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     4475..4494
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4514..4591)
                     /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 ggttacgctg ttgattatag cgaacccatc attattaaag aaaaaggaga aataaaggtt
      481 gttaaaattg gagaacttat agatgaaatt attaaaaact caaaaaacgt taggaaagat
      541 ggtattttag agatagcaag atgtaaagac gttgaagtta ttgcctttga tagtaattat
      601 aaatttaaat ttatgcctgt ttcagaggtc tcaaggcatc cagtcagtga gatgtttgag
      661 atcgttgttg agggaaataa gaaggttaga gttactggaa gccatagcgt tttcactgta
      721 aaagataatg aagtagttcc aataagagtc gatgatctaa gggttggaga tatactggtt
      781 ttagcaaaag aattgccgaa tattgaagaa aatagaaaac tcgagccgaa tattgaagaa
      841 aatagaaaac tcgaaaaact tatcaactct gatttcatat tcttaaaaat taaaaaaatt
      901 aataaggtag agccaaccaa tggatatgct tatgatttaa ccgttccaaa tgcagaaaac
      961 ttcattgctg gatttggagg atttgtatta cacaacgcct aaggtaccac aaatcctggt
     1021 gtatccgctt ggcaggtcaa cacagcttat actgcgggac aattggtcac atataacggc
     1081 aagacgtata aatgtttgca gccccacacc tccttggcag gatgggaacc atccaacgtt
     1141 cctgccttgt ggcagcttca ataagcttgc ggccgcactc gagtctggta aagaaaccgc
     1201 tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt aattaaccta
     1261 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     1321 gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac tgcttccggt
     1381 agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga
     1441 accgacgaca agctgacgac cgggtctccg caagtggcac ttttcgggga aatgtgcgcg
     1501 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     1561 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     1621 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     1681 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     1741 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     1801 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     1861 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     1921 tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca aacaggaatc
     1981 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2041 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2101 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2161 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2221 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     2281 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2341 ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat
     2401 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2461 aataaacaaa taggcatgca gcgctcttcc gcttcctcgc tcactgactc gctacgctcg
     2521 gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg gttatccaca
     2581 gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc ggaagaagcc
     2641 aacgccgcag gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2701 ctcaagccag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2761 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2821 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgttggtatc tcagttcggt
     2881 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2941 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3001 ggcagcagcc attggtaact gatttagagg actttgtctt gaagttatgc acctgttaag
     3061 gctaaactga aagaacagat tttggtgagt gcggtcctcc aacccactta ccttggttca
     3121 aagagttggt agctcagcga accttgagaa aaccaccgtt ggtagcggtg gtttttcttt
     3181 atttatgaga tgatgaatca atcggtctat caagtcaacg aacagctatt ccgttactct
     3241 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     3301 agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     3361 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     3421 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3481 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     3541 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     3601 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     3661 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     3721 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     3781 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     3841 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     3901 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     3961 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4021 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4081 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4141 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4201 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4261 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4321 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     4381 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     4441 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     4501 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     4561 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     4621 tcctgcatta ggaaat
//
