LOCUS       sequence-11499-e        3910 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 3910)
  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 3910)
  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: pHYRSF1-02
FEATURES             Location/Qualifiers
     source          1..3910
                     /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             464..508
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(546..564)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      560..607
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(837..1652)
                     /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(837..1652)
                     /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     1560..1579
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1653..1744)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1760..2509)
                     /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(1770..1787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2010..2029)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2705..3787)
                     /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     3749..3768
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3788..3865)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg atcaaaatag ccacacgtaa atatttaggc
      181 aaacaaaatg tctatgacat tggagttgag cgcgaccata attttgcact caaaaatggc
      241 ttcatagctt ctaattgttt caatggtacc cagtacaaac ttatcctgaa cggtaaaacc
      301 ctgaaaggtg aaaccaccac cgaagctgtt gacgctgcta ccgcggaaaa agttttcaaa
      361 cagtacgcta acgacaacgg tgttgacggt gaatggacct acgacgacgc taccaaaacc
      421 tacaccgtaa cggaataagc ttgcggccgc actcgagtct ggtaaagaaa ccgctgctgc
      481 gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc
      541 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
      601 ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa
      661 taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac
      721 gacaagctga cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg cgcggaaccc
      781 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
      841 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
      901 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
      961 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1021 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1081 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     1141 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1201 tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1261 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1321 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1381 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     1441 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     1501 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     1561 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     1621 gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata ttattgaagc
     1681 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     1741 caaataggca tgcagcgctc ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt
     1801 cgactgcggc gagcggtgtc agctcactca aaagcggtaa tacggttatc cacagaatca
     1861 ggggataaag ccggaaagaa catgtgagca aaaagcaaag caccggaaga agccaacgcc
     1921 gcaggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1981 ccagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2041 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2101 ttcgggaagc gtggcgcttt ctcatagctc acgctgttgg tatctcagtt cggtgtaggt
     2161 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2221 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2281 agccattggt aactgattta gaggactttg tcttgaagtt atgcacctgt taaggctaaa
     2341 ctgaaagaac agattttggt gagtgcggtc ctccaaccca cttaccttgg ttcaaagagt
     2401 tggtagctca gcgaaccttg agaaaaccac cgttggtagc ggtggttttt ctttatttat
     2461 gagatgatga atcaatcggt ctatcaagtc aacgaacagc tattccgtta ctctagattt
     2521 cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt
     2581 aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     2641 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     2701 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2761 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     2821 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     2881 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     2941 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     3001 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     3061 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     3121 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     3181 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     3241 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     3301 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     3361 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     3421 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     3481 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     3541 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     3601 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     3661 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     3721 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     3781 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     3841 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     3901 attaggaaat
//
