LOCUS       sequence-229637-        4518 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 4518)
  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 4518)
  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: pBHDuet45
FEATURES             Location/Qualifiers
     source          1..4518
                     /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             1072..1116
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1154..1172)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1168..1215
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1445..2260)
                     /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(1445..2260)
                     /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     2168..2187
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2261..2352)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2368..3117)
                     /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(2378..2395)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2618..2637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3313..4395)
                     /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     4357..4376
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4396..4473)
                     /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 ctatagtgtt gattacaacg aaccaataat cattaaagag
      361 aatggagaaa taaaagttgt taaaattgga gaacttatag ataaaattat tgaaaactca
      421 gagaatatta gaagagaggg catcttagag atagcaaaat gtaaaggtat tgaagttatt
      481 gcctttaaca gcaattacaa atttaaattc atgcctgttt cggaggtttc aaggcatcca
      541 gttagtgaga tgtttgaaat agttgttgaa gggaataaaa aggttagagt taccagaagc
      601 catagtgtct ttaccataag agataatgag gtagttccaa taagagttga tgagctaaaa
      661 gttggagata tattagtttt agcaaaagaa ttgccgaata ccaatagaaa actcgaaaaa
      721 cttataaact ctgatttcat attcttaaaa attaaagaga ttaataaggt agagccaacc
      781 agtggatatg cctatgattt aactgttcca aatgcagaaa acttcgttgc tggatttgga
      841 ggatttgtat tacacaacac catccacggt acccagtaca aacttatcct gaacggtaaa
      901 accctgaaag gtgaaaccac caccgaagct gttgacgctg ctaccgcgga aaaagttttc
      961 aaacagtacg ctaacgacaa cggtgttgac ggtgaatgga cctacgacga cgctaccaaa
     1021 acctacaccg taacggaata agcttggctg ttttggcggc tcgagtctgg taaagaaacc
     1081 gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc
     1141 taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1201 ttgaggggtt ttttgctgaa acctcaggca tttgagaagc acacggtcac actgcttccg
     1261 gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct
     1321 gaaccgacga caagctgacg accgggtctc cgcaagtggc acttttcggg gaaatgtgcg
     1381 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     1441 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     1501 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     1561 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     1621 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     1681 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     1741 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     1801 attgcgcctg agcgagacga aatacgcggt cgctgttaaa aggacaatta caaacaggaa
     1861 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     1921 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     1981 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     2041 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     2101 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     2161 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     2221 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt
     2281 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     2341 aaaataaaca aataggcatg cagcgctctt ccgcttcctc gctcactgac tcgctacgct
     2401 cggtcgttcg actgcggcga gcggtgtcag ctcactcaaa agcggtaata cggttatcca
     2461 cagaatcagg ggataaagcc ggaaagaaca tgtgagcaaa aagcaaagca ccggaagaag
     2521 ccaacgccgc aggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2581 cgctcaagcc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2641 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2701 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgttggta tctcagttcg
     2761 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2821 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2881 ctggcagcag ccattggtaa ctgatttaga ggactttgtc ttgaagttat gcacctgtta
     2941 aggctaaact gaaagaacag attttggtga gtgcggtcct ccaacccact taccttggtt
     3001 caaagagttg gtagctcagc gaaccttgag aaaaccaccg ttggtagcgg tggtttttct
     3061 ttatttatga gatgatgaat caatcggtct atcaagtcaa cgaacagcta ttccgttact
     3121 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
     3181 taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     3241 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     3301 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3361 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     3421 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     3481 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     3541 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     3601 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     3661 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     3721 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     3781 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     3841 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     3901 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     3961 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     4021 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     4081 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     4141 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     4201 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     4261 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     4321 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     4381 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     4441 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     4501 actcctgcat taggaaat
//
