LOCUS       sequence-411030-        5829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ISCbt2 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5829)
  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 5829)
  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_ISCbt2_reRNA
FEATURES             Location/Qualifiers
     source          1..5829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(69..88)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      164..709
                     /direction=RIGHT
                     /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     610..629
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     826..843
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1235..1337
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1338..1997
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1404..1423)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(2553..3008)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2690..2711)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2902..2921
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        complement(3466..3557)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(3576..3603)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3695..3781)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3934..3951
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     promoter        complement(4216..4250)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     RBS             5746..5757
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(5773..5807)
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
ORIGIN
        1 tgagcagatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
       61 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
      121 catgagccta gaaatatttt atctgattaa taagatgatc ttcttgagat cgttttggtc
      181 tgcgcgtaat ctcttgctct gaaaacgaaa aaaccgcctt gcagggcggt ttttcgaagg
      241 ttctctgagc taccaactct ttgaaccgag gtaactggct tggaggagcg cagtcaccaa
      301 aacttgtcct ttcagtttag ccttaaccgg cgcatgactt caagactaac tcctctaaat
      361 caattaccag tggctgctgc cagtggtgct tttgcatgtc tttccgggtt ggactcaaga
      421 cgatagttac cggataaggc gcagcggtcg gactgaacgg ggggttcgtg catacagtcc
      481 agcttggagc gaactgccta cccggaactg agtgtcaggc gtggaatgag acaaacgcgg
      541 ccataacagc ggaatgacac cggtaaaccg aaaggcagga acaggagagc gcacgaggga
      601 gccgccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc accactgatt
      661 tgagcgtcag atttcgtgat gcttgtcagg ggggcggagc ctatggaaaa acggctttgc
      721 cgcggccctc tcacttccct gttaagtatc ttcctggcat cttccaggaa atctccgccc
      781 cgttcgtaag ccatttccgc tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc
      841 gaggaagcgg aatatatcct gtatcacata ttctgctgac gcaccggtgc agcctttttt
      901 ctcctgccac atgaagcact tcactgacac cctcatcagt gccaacatag taagccagta
      961 tacactccgc tagcgctgat gtccggcggt gcttttgccg ttacgcacca ccccgtcagt
     1021 agctgaacag gagggacagc tgatagaaac agaagccact ggagcacctc aaaaacacca
     1081 tcatacacta aatcagtaag ttggcagcat cacccgacgc actttgcgcc gaataaatac
     1141 ctgtgacgga agatcacttc gcagaataaa taaatcctgg tgtccctgtt gataccggga
     1201 agccctgggc caacttttgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact
     1261 ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag
     1321 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
     1381 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
     1441 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
     1501 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gagttccgta
     1561 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
     1621 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
     1681 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
     1741 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
     1801 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     1861 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     1921 tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     1981 ggcagggcgg ggcgtaattt ttttaaggca gttattggtg cccttaaacg cctggttgct
     2041 acgcctgaat aagtgataat aagcggatga atggcagaaa ttcgaaagca aattcgaccc
     2101 ggtcgtcggt tcagggcagg gtcgttaaat agccgcttat gtctattgct ggtttaccgg
     2161 tttattgact accggaagca gtgtgaccgt gtgcttctca aatgcctgac tggtaacagg
     2221 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2281 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2341 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2401 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2461 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2521 ttatcaaaaa ggatcttcac ctagatcctt ttaaattgta aacgttaata ttttgttaaa
     2581 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2641 aatcccttat aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa
     2701 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2761 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
     2821 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2881 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2941 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     3001 gggcgcgtaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3061 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3121 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3181 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3241 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3301 gttgccggga agctagagta agtagttcgc cagttaatag tttgctgcac ccaactgatc
     3361 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3421 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3481 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3541 ttagaaaaat aaacaaaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct
     3601 tctgcttaat ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
     3661 gttgcttcgc aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
     3721 ccgacaaaca acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt
     3781 gatgcctggc agttccctac tctcgcatgg ggagacccca cactaccatc ggcgctacgg
     3841 cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc
     3901 aaattctgtt ttatcagacc gcttctgcgt tctgatttaa tctgtatcag gctgaaaatc
     3961 ttctctcatc cgccaaaaca gccaagctta aaaaacgcaa tgatgatctc cgagcctgaa
     4021 tactctgcac aagctaaagc ttggcaggtt ctacttctat caattctctt tttaactctc
     4081 gcttaattat aaaattaagt tacaacattc aaactattga attgattatc tatagacttt
     4141 tttgccaata gttgcaacca actaattaac gacaaaggca cgattctaat accttatttt
     4201 atatttatat tcccaactag tattatacct aggactgagc tagctgtcaa agatctctca
     4261 ggatcctatt cccatactat ttaataatct atttttacaa tttttcaatc ttaatatttc
     4321 tatatcatgc aattttttaa aactatcaaa ttcatctaat aatttttctt tatttacttt
     4381 atctaattta agttttttat ctataataac atttttaata attaaagcac tcattaaatc
     4441 tctttgtatt tgaatttctt gctcattaat tttaaaccta ttccatcttt cacttaaaga
     4501 cttcttaata tattcatttg taaagggatt atattgactt gcctttattt tataagtatc
     4561 tattttaaac aaccctaaat tatgatattt gagtttccta ttaattatat caagcaacat
     4621 actaggtgct ttgtttgcta gactcttacc aaatcttttc tttttattga atctacctgt
     4681 tttctcattt atagtagttt cttttgctct agcttgtaac cccttataat tcatagtttc
     4741 tactttaatg atgctaccta atgataatat ataattagcc attttttcat ggtcttgctt
     4801 tcttatactg gctaatctac tttgtatatc tcttaacttt gctttggttt ttaaataatt
     4861 attactatta atccatctat ctctatttcc ctttttaata gttccatctt cattatactt
     4921 attaggatta ttagcacgtc tttgtctatc taatttccta tttaatattc ttttttgatt
     4981 ttctatatta tcaatctcag gtgctaactc taataactta acatcatttt cactagatac
     5041 tgcaatagtt cttgtaccta tatccaaacc tacctcacct gttcctaatg tatgttttat
     5101 aggtggtatc ccttctaata ttagttgaac ataatatcta attgaattac ctttgtattt
     5161 tttaattatt ctacaaaatt taactctatc ttgaatgcat ttttgagcat aagtatcatt
     5221 ttttcttata attacaggca attttaattt attaaagtat ataaatccat ctctatatct
     5281 tattccttgt ctattattaa gtccttccat agaatacatt tcattatatt taataaaatt
     5341 aactctttta gcttcaccaa atctcattcg atcaatagat ttaatcgctc ttgccgataa
     5401 tgcttgtact gtattagcat ctatattttt aaattcattt acccttaatg aggtggaata
     5461 tttgtttaaa tcaaaattac taaatttata ttttaattgt acttctttga atagtttatt
     5521 tttattaatc ttcttagttt cttttcttgc ttgttgatac attttagatt ctctcattaa
     5581 tctaaatctt tttaatcctt cacctaataa agcattatac aactttcttg ataattcaaa
     5641 gtatgtattt aacgcactta tatcagattt agtagcccct aatctcaaag ttaaaacata
     5701 actaggagtt tttgatttag ccacagtatt catgaattcg aattctttct cctctttaga
     5761 tcttttcccg gggctagcat tatacctagg actgagctag ctgtcagatc gatgataagc
     5821 tgtcaaaca
//
