LOCUS       sequence-41143-e        4102 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 4102)
  AUTHORS   Muona M, Aranko AS, Raulinaitis V, Iwai H
  TITLE     Segmental isotopic labeling of multi-domain and fusion proteins by 
            protein trans-splicing in vivo and in vitro.
  JOURNAL   Nat Protoc. 2010  . 5(3):574-87.
  PUBMED    20203672
REFERENCE   2  (bases 1 to 4102)
  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: "Nat Protoc.
            2010  . 5(3):574-87."
COMMENT     Sequence Label: pHYRSF1
FEATURES             Location/Qualifiers
     source          1..4102
                     /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             656..700
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(738..756)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      752..799
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1029..1844)
                     /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(1029..1844)
                     /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     1752..1771
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1845..1936)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1952..2701)
                     /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(1962..1979)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2202..2221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2897..3979)
                     /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     3941..3960
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3980..4057)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg cagtacaaac ttatcctgaa cggtaaaacc
      181 ctgaaaggtg aaaccaccac cgaagctgtt gacgctgcta ccgcggaaaa agttttcaaa
      241 cagtacgcta acgacaacgg tgttgacggt gaatggacct acgacgacgc taccaaaacc
      301 tacaccgtaa cggaaggatc ctgtttaagc tatgaaacgg aaatattgac agtagaatat
      361 ggattattac cgattggtaa aattgtagaa aagcgcatcg aatgtactgt ttatagcgtt
      421 gataataatg gaaatattta tacacaacct gtagcacaat ggcacgatcg cggagaacaa
      481 gaggtgtttg agtattgttt ggaagatggt tcattgattc gggcaacaaa agaccataag
      541 tttatgactg ttgatggtca aatgttgcca attgatgaaa tatttgaacg tgaattggat
      601 ttgatgcggg ttgataattt gccgaattaa gcttgcggcc gcactcgagt ctggtaaaga
      661 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
      721 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      781 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
      841 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
      901 ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg
      961 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1021 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     1081 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     1141 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     1201 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     1261 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     1321 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     1381 cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca
     1441 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     1501 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     1561 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     1621 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     1681 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     1741 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     1801 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa
     1861 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     1921 tagaaaaata aacaaatagg catgcagcgc tcttccgctt cctcgctcac tgactcgcta
     1981 cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact caaaagcggt aatacggtta
     2041 tccacagaat caggggataa agccggaaag aacatgtgag caaaaagcaa agcaccggaa
     2101 gaagccaacg ccgcaggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2161 tcgacgctca agccagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2221 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2281 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt ggtatctcag
     2341 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2401 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2461 gccactggca gcagccattg gtaactgatt tagaggactt tgtcttgaag ttatgcacct
     2521 gttaaggcta aactgaaaga acagattttg gtgagtgcgg tcctccaacc cacttacctt
     2581 ggttcaaaga gttggtagct cagcgaacct tgagaaaacc accgttggta gcggtggttt
     2641 ttctttattt atgagatgat gaatcaatcg gtctatcaag tcaacgaaca gctattccgt
     2701 tactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     2761 gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg
     2821 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     2881 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2941 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     3001 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     3061 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     3121 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     3181 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     3241 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     3301 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     3361 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     3421 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     3481 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     3541 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     3601 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     3661 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     3721 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     3781 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     3841 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     3901 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     3961 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     4021 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     4081 tgcgactcct gcattaggaa at
//
