LOCUS       sequence-229635-        4989 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  analysis of cis-splicing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4989)
  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 4989)
  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: pBHDuet33
FEATURES             Location/Qualifiers
     source          1..4989
                     /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             1543..1587
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1625..1643)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1639..1686
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1916..2731)
                     /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(1916..2731)
                     /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     2639..2658
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2732..2823)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2839..3588)
                     /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(2849..2866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3089..3108)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3784..4866)
                     /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     4828..4847
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4867..4944)
                     /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 ttgaagaaga gaatgcaata
      721 gataaaaaat tcgctaaaat actggcttat atagtttctg agggttatta taatgaagaa
      781 aaacttatat tttcatttaa ttgcaataaa agagaggtta tagatgaagt aatctcctat
      841 tttaaatcat taaagtccga aataagtatc tataataaaa actcagatat acaaattgaa
      901 gtgaaagata aagaaattat caatatattg aaaaaattag gaattgagaa taaaagagtt
      961 ccttctataa tctttaaatc tccttatggg ataaaaaaat cattcataga tggattattc
     1021 aacggtaagg atacgaaaat atttacatca aaagagttag ccgaagatgc aatattttta
     1081 cttttacaga taaaagaaaa cgccatcctt aataaaaaaa tcataaaagg cattagcgtt
     1141 tatgaagtaa agagaatacc caatatatat aacaatagaa aactcgaaaa acttatcaac
     1201 tctgatttca tattcttaaa aattaaaaaa attaataagg tagagccaac caatggatat
     1261 gcttatgatt taaccgttcc aaatgcagaa aacttcattg ctggatttgg aggatttgtt
     1321 ttacacaaca ccatccacgg tacccagtac aaacttatcc tgaacggtaa aaccctgaaa
     1381 ggtgaaacca ccaccgaagc tgttgacgct gctaccgcgg aaaaagtttt caaacagtac
     1441 gctaacgaca acggtgttga cggtgaatgg acctacgacg acgctaccaa aacctacacc
     1501 gtaacggaat aagcttggct gttttggcgg ctcgagtctg gtaaagaaac cgctgctgcg
     1561 aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct
     1621 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1681 tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc ggtagtcaat
     1741 aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc tgaaccgacg
     1801 acaagctgac gaccgggtct ccgcaagtgg cacttttcgg ggaaatgtgc gcggaacccc
     1861 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     1921 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1981 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2041 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2101 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2161 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2221 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2281 gagcgagacg aaatacgcgg tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2341 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2401 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2461 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2521 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2581 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     2641 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2701 agcaagacgt ttcccgttga atatggctca tactcttcct ttttcaatat tattgaagca
     2761 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     2821 aaataggcat gcagcgctct tccgcttcct cgctcactga ctcgctacgc tcggtcgttc
     2881 gactgcggcg agcggtgtca gctcactcaa aagcggtaat acggttatcc acagaatcag
     2941 gggataaagc cggaaagaac atgtgagcaa aaagcaaagc accggaagaa gccaacgccg
     3001 caggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagc
     3061 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3121 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3181 tcgggaagcg tggcgctttc tcatagctca cgctgttggt atctcagttc ggtgtaggtc
     3241 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3301 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3361 gccattggta actgatttag aggactttgt cttgaagtta tgcacctgtt aaggctaaac
     3421 tgaaagaaca gattttggtg agtgcggtcc tccaacccac ttaccttggt tcaaagagtt
     3481 ggtagctcag cgaaccttga gaaaaccacc gttggtagcg gtggtttttc tttatttatg
     3541 agatgatgaa tcaatcggtc tatcaagtca acgaacagct attccgttac tctagatttc
     3601 agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta
     3661 attctcatgt tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     3721 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     3781 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3841 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     3901 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     3961 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4021 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg
     4081 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     4141 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     4201 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     4261 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     4321 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     4381 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     4441 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     4501 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     4561 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     4621 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     4681 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     4741 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     4801 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     4861 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     4921 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     4981 ttaggaaat
//
