LOCUS       sequence-63860-e        4419 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 4419)
  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 4419)
  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: pMMDuet19
FEATURES             Location/Qualifiers
     source          1..4419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    80..104
                     /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             160..177
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             187..204
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1033..1077
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1115..1133)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1129..1176
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1406..2221)
                     /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(1406..2221)
                     /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     2129..2148
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2222..2313)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2329..3078)
                     /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(2339..2356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2579..2598)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3274..4356)
                     /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     4318..4337
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(4357..4419,1..15))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat
       61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
      121 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      181 agcggcctgg tgccgcgcgg cagcctagga cagtacaaac ttatcctgaa cggtaaaacc
      241 ctgaaaggtg aaaccaccac cgaagctgtt gacgctgcta ccgcggaaaa agttttcaaa
      301 cagtacgcta acgacaacgg tgttgacggt gaatggacct acgacgacgc taccaaaacc
      361 tacaccgtaa cggaaggatc cggttgttta gcaggcgata gtctggtgac attagtagat
      421 agcggactgc aagtgccaat taaggaatta gtaggtaaat ctggttttgc agtttgggca
      481 ttgaacgaag ctacaatgca gctagaaaag gcaattgtta gcaatgcttt ttcgactggt
      541 ataaagcctc tgtttacttt gacaactcga ttggggcgga aaatccgtgc cacaggtaat
      601 cacaagtttt taacaattaa tggctggaaa agacttgatg aattaactcc taaagaacat
      661 ctcgctttac caagaaatct ccccagttct ggtactcttg ccaaaagtga tatttattgg
      721 gatgaaatag tttcaattga atatagtggt gaggaagaag tgtttgacct caccgttcct
      781 ggtttgcata attttgttgc aaataatatt attgttcaca attccattgg tacccagtac
      841 aaacttatcc tgaacggtaa aaccctgaaa ggtgaaacca ccaccgaagc tgttgacgct
      901 gctaccgcgg aaaaagtttt caaacagtac gctaacgaca acggtgttga cggtgaatgg
      961 acctacgacg acgctaccaa aacctacacc gtaacggaat aagcttggct gttttggcgg
     1021 ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct
     1081 actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc
     1141 cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag
     1201 cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg
     1261 gctatttaac gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg
     1321 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1381 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     1441 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1501 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     1561 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1621 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     1681 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     1741 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa
     1801 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1861 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     1921 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1981 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     2041 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     2101 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     2161 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     2221 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2281 acatatttga atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct
     2341 cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa
     2401 aagcggtaat acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa
     2461 aaagcaaagc accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct
     2521 gacgagcatc acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa
     2581 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2641 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2701 cgctgttggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2761 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2821 gtaagacacg acttatcgcc actggcagca gccattggta actgatttag aggactttgt
     2881 cttgaagtta tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc
     2941 tccaacccac ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc
     3001 gttggtagcg gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca
     3061 acgaacagct attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc
     3121 tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac
     3181 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3241 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3301 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3361 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3421 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3481 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3541 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3601 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3661 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3721 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3781 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3841 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     3901 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     3961 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4021 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4081 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4141 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4201 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4261 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4321 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4381 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcg
//
