LOCUS       sequence-418005-        5497 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ISNsp3 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5497)
  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 5497)
  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_ISNsp3_reRNA
FEATURES             Location/Qualifiers
     source          1..5497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(24..126)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      complement(652..1197)
                     /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(732..751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1273..1292
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1383..1417
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     RBS             1433..1444
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        2589..2623
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(2907..2924)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3077..3163
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3255..3282
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3301..3392
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3850..4305
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3937..3956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4147..4168
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4861..5497,1..23))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5435..5454
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca
       61 aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc
      121 cgatcaacgt ctcattttcg ccaaaagttg gcccagggct tcccggtatc aacagggaca
      181 ccaggattta tttattctgc gaagtgatct tccgtcacag gtatttattc ggcgcaaagt
      241 gcgtcgggtg atgctgccaa cttactgatt tagtgtatga tggtgttttt gaggtgctcc
      301 agtggcttct gtttctatca gctgtccctc ctgttcagct actgacgggg tggtgcgtaa
      361 cggcaaaagc accgccggac atcagcgcta gcggagtgta tactggctta ctatgttggc
      421 actgatgagg gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc
      481 gtcagcagaa tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct
      541 cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag
      601 atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag
      661 gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc
      721 gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt
      781 tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac
      841 gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc
      901 ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa
      961 gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg
     1021 aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca
     1081 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc
     1141 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa
     1201 gatcatctta ttaatcagat aaaatatttc taggctcatg agcccgaagt ggcgagcccg
     1261 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     1321 gatgccggcc acgatgcgtc cggcgtagag gatctgctca tgtttgacag cttatcatcg
     1381 atctgacagc tagctcagtc ctaggtataa tgctagcccc gggaaaagat ctaaagagga
     1441 gaaagaattc atgaaaacct tgaagttcaa actgtaccag cacaaaagaa atagattcct
     1501 caagcgcatg attaatgcga gtggggtaat ctacaaccat tgtattgccc tacacaaacg
     1561 ctactaccga atgtggggca aacacttaag ttgtgcaaaa cttcaagctc acattgctaa
     1621 actgcggaag cgtaacccat tttggcaaac tgtaggatct caggcagtgc aagatatctg
     1681 tcaacgcatt gaaaaagctt accaattatt ttttaaacac aataagaagg gggttagacc
     1741 accaggattt aagaaggtga agaaatataa atccttcacc ctaaaacaag caggttataa
     1801 gtttttaggt ggcaacagag taaaaatcgg taatcgagtt tatcaattct ggaagtcgag
     1861 acagatagag ggaacagtta aaaccctaac tataaagcgc acaccgttag gtgagttgtt
     1921 cgtggttgtg gtagttgacg acggtattaa gccagaaatc gagattaaga cgggtaaaat
     1981 cgctggtttt gactttggtt tgaagacatt tctcacttgc tcagacggca caaaaattga
     2041 ttctcctcag tttttcaaac aatctctaaa cgctatcacc aaagctagta gacaacactc
     2101 aaagaaactc aaaggttcgg ctaatcggaa acgagcgaga cttaatctag tacgcaagta
     2161 cgaagatatt tctaatcgcc gtcgtgactg gttctggaaa ctagcttatg agttaacaga
     2221 taagtttgat gttctctgct ttgaaacctt aaacctcaag ggaatgcaac gacgttgggg
     2281 caggaaaata tcagatttgg cgtttggtga gtttctggaa attctagaat gggttgccaa
     2341 aaagaagaat aaactgattg tcttcattga tcagtggtat cccagcacta agacctgttc
     2401 tcactgtggt catgtcttag agagtattga tttgtctgtt cggtcgtgga gttgtccgtc
     2461 ctgccagtca gtgaacgaga gagacgaaaa cgcctctaag gtaatttgtg cagtcggggc
     2521 atcgactgtt gggttaggtg atgtcagact ggctacgcca gcaatcgctg ttggatcctg
     2581 agagatcttt gacagctagc tcagtcctag gtataatact agtgttctca ctgtggtcat
     2641 gtcttagaga gtattgattt gtctgttcgg tcgtggagtt gtccgtcctg ccagtcagtg
     2701 aacgagagag acgaaaacgc ctctaaggta atttgtgcag tcggggcatc gactgttggg
     2761 ttaggtgatg tcagactggc tacgccagca atcgctgttt gagcctagaa tcccccgaat
     2821 tatattcggg ggagtatgtc aagctcggag atcatcattg cgttttttaa gcttggctgt
     2881 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     2941 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     3001 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     3061 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     3121 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     3181 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     3241 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
     3301 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3361 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     3421 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     3481 aagtaaaaga tgctgaagat cagttgggtg cagcaaacta ttaactggcg aactacttac
     3541 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3601 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3661 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3721 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3781 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     3841 gattgattta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3901 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3961 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4021 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4081 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4141 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
     4201 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4261 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttaaaag gatctaggtg
     4321 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4381 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4441 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4501 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4561 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4621 tacctcgctc tgctaatcct gttaccagtc aggcatttga gaagcacacg gtcacactgc
     4681 ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct
     4741 gccctgaacc gacgaccggg tcgaatttgc tttcgaattt ctgccattca tccgcttatt
     4801 atcacttatt caggcgtagc aaccaggcgt ttaagggcac caataactgc cttaaaaaaa
     4861 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
     4921 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
     4981 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
     5041 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
     5101 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
     5161 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
     5221 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
     5281 accgtctttc attgccatac ggaactccgg atgagcattc atcaggcggg caagaatgtg
     5341 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     5401 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     5461 ttctttacga tgccattggg atatatcaac ggtggta
//
