LOCUS       sequence-229638-        4560 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 4560)
  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 4560)
  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: pBHDuet64
FEATURES             Location/Qualifiers
     source          1..4560
                     /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             1114..1158
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1196..1214)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1210..1257
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1487..2302)
                     /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(1487..2302)
                     /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     2210..2229
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2303..2394)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2410..3159)
                     /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(2420..2437)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2660..2679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3355..4437)
                     /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     4399..4418
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4438..4515)
                     /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 gaatgaagta
      721 aagagaatac ccaatatata taacaataga aaactcgaaa aacttatcaa ctctgatttc
      781 atattcttaa aaattaaaaa aattaataag gtagagccaa ccaatggata tgcttatgat
      841 ttaaccgttc caaatgcaga aaacttcatt gctggatttg gaggatttgt tttacacaac
      901 accatccacg gtacccagta caaacttatc ctgaacggta aaaccctgaa aggtgaaacc
      961 accaccgaag ctgttgacgc tgctaccgcg gaaaaagttt tcaaacagta cgctaacgac
     1021 aacggtgttg acggtgaatg gacctacgac gacgctacca aaacctacac cgtaacggaa
     1081 taagcttggc tgttttggcg gctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa
     1141 cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct
     1201 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1261 aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
     1321 aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gacaagctga
     1381 cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1441 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     1501 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1561 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1621 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1681 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     1741 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     1801 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     1861 gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     1921 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     1981 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     2041 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     2101 catctgtaac atcattggca acactacctt tgccatgttt cagaaacaac tctggcgcat
     2161 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     2221 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     2281 tttcccgttg aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg
     2341 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggca
     2401 tgcagcgctc ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc
     2461 gagcggtgtc agctcactca aaagcggtaa tacggttatc cacagaatca ggggataaag
     2521 ccggaaagaa catgtgagca aaaagcaaag caccggaaga agccaacgcc gcaggcgttt
     2581 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag ccagaggtgg
     2641 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2701 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2761 gtggcgcttt ctcatagctc acgctgttgg tatctcagtt cggtgtaggt cgttcgctcc
     2821 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2881 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccattggt
     2941 aactgattta gaggactttg tcttgaagtt atgcacctgt taaggctaaa ctgaaagaac
     3001 agattttggt gagtgcggtc ctccaaccca cttaccttgg ttcaaagagt tggtagctca
     3061 gcgaaccttg agaaaaccac cgttggtagc ggtggttttt ctttatttat gagatgatga
     3121 atcaatcggt ctatcaagtc aacgaacagc tattccgtta ctctagattt cagtgcaatt
     3181 tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg
     3241 ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     3301 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     3361 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3421 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     3481 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     3541 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     3601 gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga
     3661 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     3721 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     3781 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     3841 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     3901 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     3961 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     4021 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     4081 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     4141 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     4201 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     4261 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     4321 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     4381 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     4441 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     4501 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat
//
