LOCUS       sequence-62154-e        4392 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 4392)
  AUTHORS   Oeemig JS, Aranko AS, Djupsjobacka J, Heinamaki K, Iwai H
  TITLE     Solution structure of DnaE intein from Nostoc punctiforme: 
            structural basis for the design of a new split intein suitable for 
            site-specific chemical modification.
  JOURNAL   FEBS Lett. 2009 May 6;583(9):1451-6. doi: 
            10.1016/j.febslet.2009.03.058. Epub 2009 Apr 1.
  PUBMED    19344715
REFERENCE   2  (bases 1 to 4392)
  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.febslet.2009.03.058"; journalName: "FEBS Lett"; date: 
            "2009-05-6- 6"; volume: "583"; issue: "9"; pages: "1451-6"
COMMENT     Sequence Label: pHYDuet93
FEATURES             Location/Qualifiers
     source          1..4392
                     /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             1006..1050
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1088..1106)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1102..1149
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1379..2194)
                     /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(1379..2194)
                     /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     2102..2121
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2195..2286)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2302..3051)
                     /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(2312..2329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2552..2571)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3247..4329)
                     /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     4291..4310
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(4330..4392,1..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 ctgtttaagc tatgaaacgg aaatattgac agtagaatat
      421 ggattattac cgattggtaa aattgtagaa aagcgcatcg aatgtactgt ttatagcgtt
      481 gataataatg gaaatattta tacacaacct gtagcacaat ggcacgatcg cggagaacaa
      541 gaggtgtttg agtattgttt ggaagatggt tcattgattc gggcaacaaa agaccataag
      601 tttatgactg ttgatggtca aatgttgcca attgatgaaa tatttgaacg tgaattggat
      661 ttgatgcggg ttgataattt gccgaatatc aaaatagcca cacgtaaata tttaggcaaa
      721 caaaatgtct atgacattgg agttgagcgc gaccataatt ttgcactcaa aaattaagct
      781 ttagcttcta attgtttcaa tggtacccag tacaaactta tcctgaacgg taaaaccctg
      841 aaaggtgaaa ccaccaccga agctgttgac gctgctaccg cggaaaaagt tttcaaacag
      901 tacgctaacg acaacggtgt tgacggtgaa tggacctacg acgacgctac caaaacctac
      961 accgtaacgg aataagcttg gctgttttgg cggctcgagt ctggtaaaga aaccgctgct
     1021 gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt aacctaggct
     1081 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1141 ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct tccggtagtc
     1201 aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg
     1261 acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg tgcgcggaac
     1321 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1381 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1441 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1501 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1561 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1621 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     1681 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1741 cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca ggaatcgaat
     1801 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1861 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1921 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1981 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     2041 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2101 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2161 tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa tattattgaa
     2221 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2281 aacaaatagg catgcagcgc tcttccgctt cctcgctcac tgactcgcta cgctcggtcg
     2341 ttcgactgcg gcgagcggtg tcagctcact caaaagcggt aatacggtta tccacagaat
     2401 caggggataa agccggaaag aacatgtgag caaaaagcaa agcaccggaa gaagccaacg
     2461 ccgcaggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2521 agccagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2581 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2641 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt ggtatctcag ttcggtgtag
     2701 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2761 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2821 gcagccattg gtaactgatt tagaggactt tgtcttgaag ttatgcacct gttaaggcta
     2881 aactgaaaga acagattttg gtgagtgcgg tcctccaacc cacttacctt ggttcaaaga
     2941 gttggtagct cagcgaacct tgagaaaacc accgttggta gcggtggttt ttctttattt
     3001 atgagatgat gaatcaatcg gtctatcaag tcaacgaaca gctattccgt tactctagat
     3061 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
     3121 gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     3181 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     3241 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3301 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     3361 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     3421 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     3481 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
     3541 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     3601 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     3661 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     3721 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     3781 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     3841 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     3901 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     3961 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     4021 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     4081 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     4141 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     4201 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     4261 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     4321 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     4381 aaaggttttg cg
//
