LOCUS       sequence-229634-        4554 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 4554)
  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 4554)
  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: pBHDuet50F
FEATURES             Location/Qualifiers
     source          1..4554
                     /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             1108..1152
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1190..1208)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1204..1251
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1481..2296)
                     /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(1481..2296)
                     /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     2204..2223
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2297..2388)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2404..3153)
                     /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(2414..2431)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2654..2673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3349..4431)
                     /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     4393..4412
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4432..4509)
                     /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 gagccgaata ttgaagaaaa tagaaaactc gaaaaactta tcaactctga tttcatattc
      781 ttaaaaatta aaaaaattaa taaggtagag ccaaccaatg gatatgctta tgatttaacc
      841 gttccaaatg cagaaaactt cattgctgga tttggaggat ttgttttaca caacaccatc
      901 cacggtaccc agtacaaact tatcctgaac ggtaaaaccc tgaaaggtga aaccaccacc
      961 gaagctgttg acgctgctac cgcggaaaaa gttttcaaac agtacgctaa cgacaacggt
     1021 gttgacggtg aatggaccta cgacgacgct accaaaacct acaccgtaac ggaataagct
     1081 tggctgtttt ggcggctcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag
     1141 cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa
     1201 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaacct
     1261 caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag
     1321 caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgacaag ctgacgaccg
     1381 ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1441 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1501 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1561 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1621 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1681 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1741 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1801 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1861 cgcggtcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1921 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     1981 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2041 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2101 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2161 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2221 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2281 gttgaatatg gctcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2341 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggcatgcagc
     2401 gctcttccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg
     2461 tgtcagctca ctcaaaagcg gtaatacggt tatccacaga atcaggggat aaagccggaa
     2521 agaacatgtg agcaaaaagc aaagcaccgg aagaagccaa cgccgcaggc gtttttccat
     2581 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagccagag gtggcgaaac
     2641 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2701 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2761 ctttctcata gctcacgctg ttggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2821 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2881 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccat tggtaactga
     2941 tttagaggac tttgtcttga agttatgcac ctgttaaggc taaactgaaa gaacagattt
     3001 tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa gagttggtag ctcagcgaac
     3061 cttgagaaaa ccaccgttgg tagcggtggt ttttctttat ttatgagatg atgaatcaat
     3121 cggtctatca agtcaacgaa cagctattcc gttactctag atttcagtgc aatttatctc
     3181 ttcaaatgta gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc
     3241 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     3301 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     3361 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3421 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     3481 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     3541 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     3601 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     3661 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3721 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3781 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     3841 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     3901 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     3961 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     4021 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     4081 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     4141 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     4201 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     4261 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     4321 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     4381 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     4441 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     4501 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aaat
//
