LOCUS       sequence-460756-        7065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SisTnpB1 and RNP complex in E. coli Rosetta (DE3) cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7065)
  AUTHORS   Xu Y, Yin W, Cheng Y, Zeng W, Li W, Chen W, Wang F, Peng N, Ma L, 
            Liu T
  TITLE     Collateral nuclease activity of TnpB triggered by high temperature 
            enables fast and sensitive nucleic acid detection.
  JOURNAL   Commun Biol. 2024 Nov 20;7(1):1541. doi: 10.1038/s42003-024-07123-3.
  PUBMED    39567725
REFERENCE   2  (bases 1 to 7065)
  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: "Commun 
            Biol."; date: "2024-11-20"; volume: "7(1):1541. doi"; pages: " 
            10.1038/s42003-024-07123-3"
COMMENT     Sequence Label: pET-SisTnpB1–omega-RNA
FEATURES             Location/Qualifiers
     source          1..7065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             32..49
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(102..120)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      116..163
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      200..655
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(287..306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     497..518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(747..1562)
                     /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(747..1562)
                     /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     1470..1489
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1554..1573
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1684..2272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2173..2192
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2426..2443
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2458..2597
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2523..2542)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2683..2705
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2699..2890)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3699..4781)
                     /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     4743..4762
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4782..4859)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5065..5084
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5172..5190
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5191..5215
                     /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)."
     RBS             5230..5252
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5263..5286
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     promoter        6727..6745
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     ncRNA           join(7023..7065,1..25)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
ORIGIN
        1 catgcttcgg catggcgaat gggacctcga gcaccaccac caccaccact gagatccggc
       61 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
      121 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
      181 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
      241 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
      301 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
      361 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
      421 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
      481 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
      541 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
      601 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
      661 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
      721 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
      781 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
      841 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
      901 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
      961 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     1021 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     1081 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     1141 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1201 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     1261 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     1321 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     1381 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     1441 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     1501 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     1561 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     1621 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1681 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1741 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1801 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     1861 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1921 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1981 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2041 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2101 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2161 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2221 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2281 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2341 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2401 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     2461 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     2521 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     2581 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     2641 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     2701 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     2761 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     2821 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     2881 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     2941 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     3001 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     3061 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     3121 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     3181 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     3241 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     3301 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     3361 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3421 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3481 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3541 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3601 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     3661 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3721 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3781 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     3841 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     3901 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     3961 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4021 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4081 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4141 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4201 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4261 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4321 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4381 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4441 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4501 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4561 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4621 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     4681 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     4741 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     4801 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     4861 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     4921 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     4981 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     5041 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     5101 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga
     5161 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     5221 agaaataatt ttgtttaact ttaagaagga gatatacata tgcatcacca tcaccaccat
     5281 catcactgga gagccaagga gaagaatgac aagacccaac tctcattcaa gtacaagatt
     5341 tacccaacga gagaagtaga ggagaaacta atcaaagtga tgcaaattga ggctaaagta
     5401 tacaatgcct tgctagacgt gataaacaac gcgaggaaag aagggaaaaa gataacacct
     5461 aaagacactc aaaacatgct gaaagagttg aaaatagaag ggaaggaact agtttactct
     5521 aaagcccttc aaatggttaa caaccaatta tggagcaaca tcaatgctct tcatgaactg
     5581 aagaagaaag ggaagaaagt ggggaaactg aggtacaaga agatcttgaa gatcatcaat
     5641 tataatcaat ctggtttcaa ggttgaggga gacaagctga tcctctcaaa gataggagag
     5701 ataaaagtac tctttcacag accgttggaa ggcgagataa aaggagttat aataaagaag
     5761 agttcaactg gttggtatgc tatctttcaa gtcgaggtgg aaaagaagcc tcttgaaaag
     5821 gctgggaaag tggttggaat agacctagga gtggataaac tagtaactac ttctgatggt
     5881 gtagtaatag agaaccccaa ggtttttgat aaggtcgaga ggaggataaa gatcttacaa
     5941 aaatcattat ctagaaagaa gaagggatct agaaactacg agaaggttag gaaaaaacta
     6001 gctaagcttc acgagcatgt gaagaacttg atgagtgatt atattcacaa ggtgacttcg
     6061 tggttagtag aagagtatga tgagatatac gtggaagatc ttgatgtaaa ggatatagtt
     6121 gaggatagtg aaagtaaagt tttgagaaag catattcttc attctgactt ttctaagttt
     6181 gtgagctatc tctcctacaa ggctgaaaga gctggtagga gggtagtgaa agtagatccg
     6241 aggaatactt ctaagacgtg tgctaggtgc ggttacgtta agaaggactt ggctttggct
     6301 gaccgtgtgt ttgtatgccc tgagtgcggt tggactgtag atcgtgacta taatgcttct
     6361 ctaaatattc ttcgtgcggg gtcgggactg cccttagagc ctgtggacag gggacctctg
     6421 ctatacattc ccttctctga gggtgtgtat agtaagtttc ttggaagaag caggaaatct
     6481 ccatcgtgag gtggagatgc cacgtccgta agggcggggt tgttcactat atcataaccg
     6541 aattttctta tgaactcttt tctgctctct tcagtcatca ttaatggtgt acattcgtat
     6601 ctaaatccaa ctgtaccctt atacttttag caggtatgct ttccttaatc acttgctcaa
     6661 cagtgcactg caaatcttca gtaactgagc aagctggtgt tatagcgtcg acaagcttgc
     6721 ggccgctaat acgactcact atagggttaa gaaggacttg actttggctg accgtgtgtt
     6781 tgtatgtcct aaatgtggtt ggactgtaga tcgtgactat aatgcttctc taaatattct
     6841 tcgtgcgggg tcgggactgc ccttagagcc tgtggacagg ggacctctgc tatacattcc
     6901 cttctcagaa ggggtgtata gtaagtttct tggaagaagc aggaaatctc catcgtgagg
     6961 tggagatgcc acgtccgtaa gggcggggtt gttcaccggt ttttattgcc tagtatgtcg
     7021 acggccggca tggtcccagc ctcctcgctg gcgccggctg ggcaa
//
