LOCUS       sequence-410883-        5562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ISKpb69 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5562)
  AUTHORS   Xiang G, Li Y, Sun J, Huo Y, Cao S, Cao Y, Guo Y, Yang L, Cai Y, 
            Zhang YE, Wang H
  TITLE     Evolutionary mining and functional characterization of TnpB 
            nucleases identify efficient miniature genome editors.
  JOURNAL   Nat Biotechnol. 2023 Jun 29. doi: 10.1038/s41587-023-01857-x.
  PUBMED    37386294
REFERENCE   2  (bases 1 to 5562)
  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 
            Biotechnol. 2023 Jun 29. doi: 10.1038/s41587-023-01857-x."
COMMENT     Sequence Label: pBad33_ISKpn69_reRNA
FEATURES             Location/Qualifiers
     source          1..5562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..50
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     RBS             66..77
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        1285..1319
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(1605..1622)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1775..1861
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1953..1980
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1999..2090
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2548..3003
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2635..2654)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2845..2866
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3559..4218)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4133..4152
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4219..4321)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4713..4730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4847..5392)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4927..4946)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5468..5487
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cagcttatca tcgatctgac agctagctca gtcctaggta taatgctagc cccgggaaaa
       61 gatctaaaga ggagaaagaa ttcatgctaa tccagaaagc gtataaatac agactcaatc
      121 cgacagatgc acaggcgcaa cgactgcgtc agatgtgcgg ctgcgcccgt tttgtctgga
      181 attacgccct ggcagagacg cagcgtattc ttgaggccgg acaaaaactc ccttccgcct
      241 ttgagctgaa taaaatgctg acaggatgga agaaattacc tgacctggcg tttttatctg
      301 atgcctacac cgacaacctt cagcagaaac tgaaagacct gcacggggca tggatacggt
      361 gctttgataa aaaactggca gcgaaagcgc ccgtgttcaa acgtaaaacc cacggcaatg
      421 attccgttcg tttcgtcaat tttgacaaat actgccagct tgagtacaga cgcgttaagt
      481 taccctcggg gctgggctgg gtgacattcc ggcagtcccg ggccgttgtg ggtaaaatca
      541 aaaacgcaac ggtcagccag cacgctggcg gctggtatat ctcgtttcag gtagaaatgg
      601 aggtcgaaca gcccgcgcac ccgtcaacgt ctgctgtcgg cctggatgcc ggtatctgca
      661 agctggtaac gctgtcagac ggcacggtgt ttgaacctgt caacagtttt aaacagaacc
      721 aaagcagact ggcaaggtta cagcgccagc ttgcccgtaa agtgaaattc agtgcgaact
      781 ggaagaagca gaaagcaaga atttcccgct tacattcgca tatcgccaac atccgccgcg
      841 attaccttca caaagtcacg acaacaatca gcaaaaacca cgcgatgatc gttattgaag
      901 atttgaaggt ttccaacatg tcgaaatcgg cagcgggtac ggtagacgag ccggggcgca
      961 acgttgcggc aaaatcaggt cttaaccggg cgatactgga tcagggctgg tacgaaatgc
     1021 gccgccagct cgaatataag cagcgatggc gcggcggtga ggtgctggca gtaaatccgg
     1081 catataccag ccagaaatgc gcgcgttgcg gtcatacagt aaaggaaaat cgtcaatcgc
     1141 aggcagtatt tgtctgcgtg gcgtgtggat acgaagcgaa cgccgacata aacggcgctc
     1201 aaaatattct cgcggcagga catgccgtgt tgtccgggat taaccccgga cgcgcccgta
     1261 aggcggcggg atcctgagag atctttgaca gctagctcag tcctaggtat aatactagtg
     1321 tctgcgtggc gtgtggatac gaagcgaacg ccgacataaa cggcgctcaa aatattctcg
     1381 cggcaggaca tgccgtgttg tccgggatta accccggacg cgcccgtaag gcggcgtaag
     1441 ccgttaagcg aggaaccggg caatgcggta acgcgttgct accctacaaa gggaatcccc
     1501 ttcctgattc acgtagtgag aaggtggggg aggatgtcaa gctcggagat catcattgcg
     1561 ttttttaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     1621 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     1681 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     1741 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     1801 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     1861 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     1921 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     1981 tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2041 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2101 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2161 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca gcaaactatt
     2221 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2281 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2341 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2401 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2461 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2521 ttactcatat atactttaga ttgatttacg cgccctgtag cggcgcatta agcgcggcgg
     2581 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2641 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2701 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2761 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2821 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2881 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2941 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3001 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3061 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3121 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3181 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3241 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3301 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtcag gcatttgaga
     3361 agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag
     3421 cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct
     3481 gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt aagggcacca
     3541 ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc
     3601 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     3661 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     3721 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     3781 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     3841 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     3901 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     3961 atcccatatc accagctcac cgtctttcat tgccatacgg aactccggat gagcattcat
     4021 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     4081 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     4141 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     4201 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa
     4261 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc
     4321 aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg
     4381 atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc
     4441 gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg
     4501 cttctgtttc tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca
     4561 aaagcaccgc cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga
     4621 tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag
     4681 cagaatatgt gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc
     4741 gttcgactgc ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc
     4801 aggaagatac ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc
     4861 gcccccctga caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag
     4921 gactataaag ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg
     4981 cctttcggtt taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg
     5041 acactcagtt ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt
     5101 cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat
     5161 gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc
     5221 atgcgccggt taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca
     5281 gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc
     5341 ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca
     5401 tcttattaat cagataaaat atttctaggc tcatgagccc gaagtggcga gcccgatctt
     5461 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     5521 cggccacgat gcgtccggcg tagaggatct gctcatgttt ga
//
