LOCUS       sequence-65390-e        5449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5449)
  AUTHORS   Ellila S, Jurvansuu JM, Iwai H
  TITLE     Evaluation and comparison of protein splicing by exogenous inteins 
            with foreign exteins in Escherichia coli.
  JOURNAL   FEBS Lett. 2011 Oct 10.
  PUBMED    22001202
REFERENCE   2  (bases 1 to 5449)
  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"; journalName: "FEBS Lett. 
            2011 Oct 10."
COMMENT     Sequence Label: pSEDuet40
FEATURES             Location/Qualifiers
     source          1..5449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     471..490
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(510..587)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     633..652
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        633..651
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    652..676
                     /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             732..749
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             759..1856
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1713..1722
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1830..1853
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2635..2679
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2717..2735)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2731..2778
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3008..3823)
                     /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(3008..3823)
                     /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     3731..3750
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3824..3915)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3931..4680)
                     /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(3941..3958)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4181..4200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4876..5449,1..509))
                     /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"
ORIGIN
        1 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
       61 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
      121 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
      181 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
      241 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
      301 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
      361 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
      421 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
      481 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
      541 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
      601 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactatag gggaattgtg
      661 agcggataac aattcccctg tagaaataat tttgtttaac tttaataagg agatatacca
      721 tgggcagcag ccatcatcat catcatcaca gcactagtaa aatcgaagaa ggtaaactgg
      781 taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg
      841 agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc
      901 cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg
      961 gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca
     1021 agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga
     1081 tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa
     1141 cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga
     1201 tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg
     1261 cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg
     1321 cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca
     1381 ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg
     1441 gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc
     1501 cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg
     1561 ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg
     1621 aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg
     1681 aggaagagtt ggcgaaagat ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtg
     1741 aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga
     1801 tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactcgcg
     1861 gatccaagtg cgtatcaggt gaaacaccag tttaccttgc ggatggcaag acaataaaaa
     1921 taaaggatct atacagttct gagagaaaaa aagaagataa cattgttgag gctggttcgg
     1981 gagaagagat aatacatcta aaagatccca ttcaaatata ttcttatgtg gacgggacca
     2041 tagtcaggag cagatcaaga cttctataca agggcaagag ctcttatctt gtgaggatag
     2101 aaactattgg cggaagatcg gtaagcgtta caccagttca caaactcttt gtccttacgg
     2161 aaaagggtat cgaagaggtt atggcctcca acctaaaggt aggcgacatg attgctgctg
     2221 tagcagaaag cgaatctgaa gcaagagact gcggaatgag cgaggaatgc gtgatggaag
     2281 cagaagttta tacgtcactt gaagcgacat tcgatagagt aaagtctata gcgtacgaga
     2341 agggtgattt tgatgtatac gatctttccg taccagaata cggcaggaac tttataggcg
     2401 gagaaggact tctcgtactt cacaacacgg ttataggtac ccagtacaaa cttatcctga
     2461 acggtaaaac cctgaaaggt gaaaccacca ccgaagctgt tgacgctgct accgcggaaa
     2521 aagttttcaa acagtacgct aacgacaacg gtgttgacgg tgaatggacc tacgacgacg
     2581 ctaccaaaac ctacaccgta acggaataag cttgcggccg cactcgagtc tggtaaagaa
     2641 accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta
     2701 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     2761 gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt
     2821 ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc
     2881 cctgaaccga cgacaagctg acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt
     2941 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     3001 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     3061 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     3121 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     3181 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     3241 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     3301 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     3361 gtgattgcgc ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag
     3421 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     3481 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     3541 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     3601 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     3661 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     3721 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     3781 atcgcggcct agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat
     3841 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3901 agaaaaataa acaaataggc atgcagcgct cttccgcttc ctcgctcact gactcgctac
     3961 gctcggtcgt tcgactgcgg cgagcggtgt cagctcactc aaaagcggta atacggttat
     4021 ccacagaatc aggggataaa gccggaaaga acatgtgagc aaaaagcaaa gcaccggaag
     4081 aagccaacgc cgcaggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4141 cgacgctcaa gccagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4201 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4261 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgttg gtatctcagt
     4321 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4381 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4441 ccactggcag cagccattgg taactgattt agaggacttt gtcttgaagt tatgcacctg
     4501 ttaaggctaa actgaaagaa cagattttgg tgagtgcggt cctccaaccc acttaccttg
     4561 gttcaaagag ttggtagctc agcgaacctt gagaaaacca ccgttggtag cggtggtttt
     4621 tctttattta tgagatgatg aatcaatcgg tctatcaagt caacgaacag ctattccgtt
     4681 actctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg
     4741 atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg
     4801 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4861 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4921 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4981 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5041 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5101 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     5161 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5221 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5281 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5341 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5401 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacg
//
