LOCUS       sequence-229639-        4524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  analysis of cis-splicing by mini inteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4524)
  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 4524)
  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: pBHDuet50K
FEATURES             Location/Qualifiers
     source          1..4524
                     /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             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1078..1122
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1160..1178)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1174..1221
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1451..2266)
                     /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(1451..2266)
                     /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     2174..2193
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2267..2358)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2374..3123)
                     /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(2384..2401)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2624..2643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3319..4401)
                     /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     4363..4382
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4402..4479)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagcctagga cagtacaaac ttatcctgaa cggtaaaacc
      181 ctgaaaggtg aaaccaccac cgaagctgtt gacgctgcta ccgcggaaaa agttttcaaa
      241 cagtacgcta acgacaacgg tgttgacggt gaatggacct acgacgacgc taccaaaacc
      301 tacaccgtaa cggaaggatc ctacagtgtt gattatagcg aacccatcat tattaaagaa
      361 aaaggagaaa taaaggttgt taaaattgga gaacttatag atgaaattat taaaaactca
      421 aaaaacgtta ggaaagatgg tattttagag atagcaagat gtaaagacgt tgaagttatt
      481 gcctttgata gtaattataa atttaaattt atgcctgttt cagaggtctc aaggcatcca
      541 gtcagtgaga tgtttgagat cgttgttgag ggaaataaga aggttagagt tactggaagc
      601 catagcgttt tcactgtaaa agataatgaa gtagttccaa taagagtcga tgatctaagg
      661 gttggagata tactggtttt agcaaaagaa ttgccgaata ttgaagaaaa tagaaaactc
      721 gaaaaactta tcaactctga tttcatattc ttaaaaatta aaaaaattaa taaggtagag
      781 ccaaccaatg gatatgctta tgatttaacc gttccaaatg cagaaaactt cattgctgga
      841 tttggaggat ttgttttaca caacaccatc cacggtaccc agtacaaact tatcctgaac
      901 ggtaaaaccc tgaaaggtga aaccaccacc gaagctgttg acgctgctac cgcggaaaaa
      961 gttttcaaac agtacgctaa cgacaacggt gttgacggtg aatggaccta cgacgacgct
     1021 accaaaacct acaccgtaac ggaataagct tggctgtttt ggcggctcga gtctggtaaa
     1081 gaaaccgctg ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa
     1141 ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1201 cgggtcttga ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg
     1261 cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc
     1321 tgccctgaac cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa
     1381 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1441 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1501 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1561 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1621 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1681 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     1741 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     1801 ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa
     1861 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     1921 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     1981 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2041 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2101 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2161 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2221 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc
     2281 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     2341 tttagaaaaa taaacaaata ggcatgcagc gctcttccgc ttcctcgctc actgactcgc
     2401 tacgctcggt cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt
     2461 tatccacaga atcaggggat aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg
     2521 aagaagccaa cgccgcaggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2581 aatcgacgct caagccagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2641 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2701 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg ttggtatctc
     2761 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2821 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2881 tcgccactgg cagcagccat tggtaactga tttagaggac tttgtcttga agttatgcac
     2941 ctgttaaggc taaactgaaa gaacagattt tggtgagtgc ggtcctccaa cccacttacc
     3001 ttggttcaaa gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt
     3061 ttttctttat ttatgagatg atgaatcaat cggtctatca agtcaacgaa cagctattcc
     3121 gttactctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat
     3181 acgatataag ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact
     3241 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     3301 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3361 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     3421 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     3481 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     3541 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     3601 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     3661 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     3721 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     3781 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     3841 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     3901 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     3961 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     4021 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     4081 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     4141 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     4201 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     4261 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     4321 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     4381 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     4441 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     4501 tatgcgactc ctgcattagg aaat
//
