LOCUS       sequence-417998-        5900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ISCbt1 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5900)
  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 5900)
  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_ISCbt1_reRNA
FEATURES             Location/Qualifiers
     source          1..5900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(88..107)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      183..728
                     /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     629..648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     845..862
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1254..1356
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1357..2016
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1423..1442)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(2572..3027)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2709..2730)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2921..2940
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        complement(3485..3576)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(3595..3622)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3714..3800)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3953..3970
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     RBS             5836..5847
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(5863..5897)
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
ORIGIN
        1 gatgataagc tgtcaaacat gagcagatcc tctacgccgg acgcatcgtg gccggcatca
       61 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
      121 gggctcgcca cttcgggctc atgagcctag aaatatttta tctgattaat aagatgatct
      181 tcttgagatc gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg
      241 cagggcggtt tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt
      301 ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc
      361 aagactaact cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct
      421 ttccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg
      481 gggttcgtgc atacagtcca gcttggagcg aactgcctac ccggaactga gtgtcaggcg
      541 tggaatgaga caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa
      601 caggagagcg cacgagggag ccgccagggg gaaacgcctg gtatctttat agtcctgtcg
      661 ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc
      721 tatggaaaaa cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc
      781 ttccaggaaa tctccgcccc gttcgtaagc catttccgct cgccgcagtc gaacgaccga
      841 gcgtagcgag tcagtgagcg aggaagcgga atatatcctg tatcacatat tctgctgacg
      901 caccggtgca gccttttttc tcctgccaca tgaagcactt cactgacacc ctcatcagtg
      961 ccaacatagt aagccagtat acactccgct agcgctgatg tccggcggtg cttttgccgt
     1021 tacgcaccac cccgtcagta gctgaacagg agggacagct gatagaaaca gaagccactg
     1081 gagcacctca aaaacaccat catacactaa atcagtaagt tggcagcatc acccgacgca
     1141 ctttgcgccg aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt
     1201 gtccctgttg ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg
     1261 cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt
     1321 gagttatcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
     1381 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
     1441 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
     1501 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
     1561 gctcatccgg agttccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
     1621 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
     1681 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
     1741 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat
     1801 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
     1861 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
     1921 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
     1981 caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc
     2041 ccttaaacgc ctggttgcta cgcctgaata agtgataata agcggatgaa tggcagaaat
     2101 tcgaaagcaa attcgacccg gtcgtcggtt cagggcaggg tcgttaaata gccgcttatg
     2161 tctattgctg gtttaccggt ttattgacta ccggaagcag tgtgaccgtg tgcttctcaa
     2221 atgcctgact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     2281 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     2341 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2401 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     2461 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     2521 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattgtaa
     2581 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2641 aataggccga aatcggcaaa atcccttata aatcaaaaga atagcccgag atagggttga
     2701 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2761 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt
     2821 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2881 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2941 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3001 cgcttaatgc gccgctacag ggcgcgtaaa tcaatctaaa gtatatatga gtaaacttgg
     3061 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3121 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3181 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3241 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3301 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3361 ttgctgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3421 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3481 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3541 gatacatatt tgaatgtatt tagaaaaata aacaaaagag tttgtagaaa cgcaaaaagg
     3601 ccatccgtca ggatggcctt ctgcttaatt tgatgcctgg cagtttatgg cgggcgtcct
     3661 gcccgccacc ctccgggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc
     3721 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
     3781 tgagcctttc gttttatttg atgcctggca gttccctact ctcgcatggg gagaccccac
     3841 actaccatcg gcgctacggc gtttcacttc tgagttcggc atggggtcag gtgggaccac
     3901 cgcgctactg ccgccaggca aattctgttt tatcagaccg cttctgcgtt ctgatttaat
     3961 ctgtatcagg ctgaaaatct tctctcatcc gccaaaacag ccaagcttaa aaaacgcaat
     4021 gatgatctcc gagcctgaca ctcccaagac taaagccttg gggttcttag atacattaac
     4081 ttcttttata ctctcattta tttaaacttc aaataaatta caacatataa atactagtta
     4141 gtttctctaa gactttcact aattatcatt ttaataaatg attaacatta gtataaggct
     4201 catgtctaaa cctatataga ttttatatcc ccatactgga tattaattta tatccatttt
     4261 tctttaattc ttttaatctt tctatctctt tgtcgtgtaa tctctattat acctaggact
     4321 gagctagctg tcaaagatct ctcaggatcc tatccccata ctggatatta atttatatcc
     4381 atttttcttt aattctttta atctttctat ctctttgtcg tgtaatctcc taaaattatc
     4441 ataagtttca atacataaat ctctatctat tttatctaat gttttaccta caacattcat
     4501 tattaaaaat gcagaataca tatctctttg aatttgaata tcttcattcc atctgtcttt
     4561 taactctttc ttacaatatt catttgtaaa atgattatat tgacttgctt tgcatttctt
     4621 agtatctatt ttgaatagtt gagtattatt ccattttagc ttattatcta acatagttaa
     4681 aaacatacta ggtgctttat tcgctagact cttaccaaat cttttctttt tattgaatct
     4741 acctgttttc tcatttatag tagtttcttt tactctagcc tgtagtccct tataattcat
     4801 ggtttctact aaaaatctat tacccaatag taacaatcta tttattagtc tataatgact
     4861 ttgttttctg atttcagctt gtttcctttg aatttcttta agtttatttt gagttttaat
     4921 atatctatta gatttgatcc atttatcttt attattccta ttaatagttc catcttcatt
     4981 atacttatta ggattattag cacgtctttg tctatctaat ttcctattta atattctttt
     5041 ttgattttct atattatcaa tctcaggtgc taactctaat aacttaacat cattttcact
     5101 agatactgca atagttcttg tacctatatc tatccccaca ttacctttac ctatcttatt
     5161 ttttatctcg cctgtagtct tattaatctt cataggaggt attccttcaa gaattaattg
     5221 aacataaaat ttgattttac cttttacaat ccttctgatg attctacagt acttaactct
     5281 attttctaat gacatttgaa catatatatc attagttttt attattacag gtatttttaa
     5341 cccattccat attaacgaat tatcttgaaa tctaatgcca gttttattag atttaccctc
     5401 aacactattt aattcaccat atcttttaaa atatactctt ttagcttgat gaaacattag
     5461 tttctgaaat gaattaaaaa ctcttgttgc tatcttttga gctgtaaaac tatctacatt
     5521 atctttaaaa tgcttttgaa ttggtttaac atatgtatga agtgaatact cagttaaatc
     5581 atatttttta tttaattcat taaatttttt actacgttct ttgtctttac tcattttaat
     5641 tatattttta tattctttcc tatagtcttt caattctctt gtatgtttat atctttttaa
     5701 tatttcacct aaacaagagt tatatatatt tctgctgatt tctagtcttt tatttaatat
     5761 atcttcttga tacttttctg tttttaattt taaagttaat atataacttg atgtttttga
     5821 tttagccatg aattctttct cctctttaga tcttttcccg gggctagcat tatacctagg
     5881 actgagctag ctgtcagatc
//
