LOCUS       sequence-229640-        4551 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 4551)
  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 4551)
  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: pBHDuet61
FEATURES             Location/Qualifiers
     source          1..4551
                     /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             1105..1149
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1187..1205)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1201..1248
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1478..2293)
                     /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(1478..2293)
                     /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     2201..2220
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2294..2385)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2401..3150)
                     /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(2411..2428)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2651..2670)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3346..4428)
                     /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     4390..4409
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4429..4506)
                     /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 ttgaagaggt aaggagaata
      721 acaaatatat ataccaatag aaaactcgaa aaacttataa actctgattt catattctta
      781 aaaattaaag agattaataa ggtagagcca accagtggat atgcctatga tttaactgtt
      841 ccaaatgcag aaaacttcgt tgctggattt ggaggatttg tattacacaa caccatccac
      901 ggtacccagt acaaacttat cctgaacggt aaaaccctga aaggtgaaac caccaccgaa
      961 gctgttgacg ctgctaccgc ggaaaaagtt ttcaaacagt acgctaacga caacggtgtt
     1021 gacggtgaat ggacctacga cgacgctacc aaaacctaca ccgtaacgga ataagcttgg
     1081 ctgttttggc ggctcgagtc tggtaaagaa accgctgctg cgaaatttga acgccagcac
     1141 atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc tgagcaataa
     1201 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaacctcag
     1261 gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa
     1321 tagacataag cggctattta acgaccctgc cctgaaccga cgacaagctg acgaccgggt
     1381 ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1441 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1501 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1561 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1621 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1681 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1741 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1801 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1861 ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1921 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1981 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2041 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2101 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2161 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2221 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2281 gaatatggct catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     2341 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggc atgcagcgct
     2401 cttccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggtgt
     2461 cagctcactc aaaagcggta atacggttat ccacagaatc aggggataaa gccggaaaga
     2521 acatgtgagc aaaaagcaaa gcaccggaag aagccaacgc cgcaggcgtt tttccatagg
     2581 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gccagaggtg gcgaaacccg
     2641 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2701 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2761 tctcatagct cacgctgttg gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2821 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2881 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccattgg taactgattt
     2941 agaggacttt gtcttgaagt tatgcacctg ttaaggctaa actgaaagaa cagattttgg
     3001 tgagtgcggt cctccaaccc acttaccttg gttcaaagag ttggtagctc agcgaacctt
     3061 gagaaaacca ccgttggtag cggtggtttt tctttattta tgagatgatg aatcaatcgg
     3121 tctatcaagt caacgaacag ctattccgtt actctagatt tcagtgcaat ttatctcttc
     3181 aaatgtagca cctgaagtca gccccatacg atataagttg taattctcat gttagtcatg
     3241 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     3301 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     3361 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3421 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     3481 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     3541 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     3601 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     3661 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     3721 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     3781 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     3841 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     3901 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     3961 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     4021 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     4081 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     4141 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     4201 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     4261 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     4321 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     4381 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     4441 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     4501 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaaa t
//
