LOCUS       sequence-220457-        4298 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 4298)
  AUTHORS   Iwai H, Zuger S, Jin J, Tam PH
  TITLE     Highly efficient protein trans-splicing by a naturally split DnaE 
            intein from Nostoc punctiforme.
  JOURNAL   FEBS Lett. 2006 Mar 20. 580(7):1853-8.
  PUBMED    16516207
REFERENCE   2  (bases 1 to 4298)
  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. 
            2006 Mar 20. 580(7):1853-8."
COMMENT     Sequence Label: pSKDuet01
FEATURES             Location/Qualifiers
     source          1..4298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     545..564
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(584..661)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        707..725
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    726..750
                     /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             806..823
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             833..850
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        1406..1424
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1425..1449
                     /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             1558..1602
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1640..1658)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1654..1701
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1931..2746)
                     /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(1931..2746)
                     /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     2654..2673
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2747..2838)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2854..3603)
                     /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(2864..2881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3104..3123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3799..4298,1..583))
                     /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 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
       61 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      121 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      181 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      241 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      301 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      361 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      421 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      481 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      541 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
      601 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
      661 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaaattaat acgactcact
      721 ataggggaat tgtgagcgga taacaattcc cctgtagaaa taattttgtt taactttaat
      781 aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc
      841 gcgcggcagc catatgcagt acaaacttat cctgaacggt aaaaccctga aaggtgaaac
      901 caccaccgaa gctgttgacg ctgctaccgc ggaaaaagtt ttcaaacagt acgctaacga
      961 caacggtgtt gacggtgaat ggacctacga cgacgctacc aaaacctaca ccgtaacgga
     1021 aggatcctgt ttaagctatg aaacggaaat attgacagta gaatatggat tattaccgat
     1081 tggtaaaatt gtagaaaagc gcatcgaatg tactgtttat agcgttgata ataatggaaa
     1141 tatttataca caacctgtag cacaatggca cgatcgcgga gaacaagagg tgtttgagta
     1201 ttgtttggaa gatggttcat tgattcgggc aacaaaagac cataagttta tgactgttga
     1261 tggtcaaatg ttgccaattg atgaaatatt tgaacgtgaa ttggatttga tgcgggttga
     1321 taatttgccg aattaagctt gcggccgcat aatgcttaag tcgaacagaa agtaatcgta
     1381 ttgtacacgg ccgcataatc gaaattaata cgactcacta taggggaatt gtgagcggat
     1441 aacaattccc catcttagta tattagttaa gtataagaag gagatataca tatggcagat
     1501 ctcaattgga tatcggccgg ccacgcgatc gctgacgtcg gtaccctcga gtctggtaaa
     1561 gaaaccgctg ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa
     1621 ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1681 cgggtcttga ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg
     1741 cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc
     1801 tgccctgaac cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa
     1861 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1921 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1981 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     2041 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     2101 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     2161 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     2221 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2281 ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa
     2341 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     2401 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     2461 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2521 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2581 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2641 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2701 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc
     2761 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     2821 tttagaaaaa taaacaaata ggcatgcagc gctcttccgc ttcctcgctc actgactcgc
     2881 tacgctcggt cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt
     2941 tatccacaga atcaggggat aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg
     3001 aagaagccaa cgccgcaggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3061 aatcgacgct caagccagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3121 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3181 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg ttggtatctc
     3241 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3301 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3361 tcgccactgg cagcagccat tggtaactga tttagaggac tttgtcttga agttatgcac
     3421 ctgttaaggc taaactgaaa gaacagattt tggtgagtgc ggtcctccaa cccacttacc
     3481 ttggttcaaa gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt
     3541 ttttctttat ttatgagatg atgaatcaat cggtctatca agtcaacgaa cagctattcc
     3601 gttactctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat
     3661 acgatataag ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact
     3721 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     3781 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3841 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     3901 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     3961 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4021 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4081 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4141 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4201 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4261 gcgagtgaga tatttatgcc agccagccag acgcagac
//
