LOCUS       sequence-410881-        5480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ISNsp2 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5480)
  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 5480)
  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_ISNsp2_reRNA
FEATURES             Location/Qualifiers
     source          1..5480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..35
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     RBS             51..62
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        1213..1247
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(1508..1525)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1678..1764
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1856..1883
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1902..1993
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2451..2906
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2538..2557)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2748..2769
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3462..4121)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4036..4055
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4122..4224)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4616..4633)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4750..5295)
                     /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(4830..4849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5371..5390
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ctgacagcta gctcagtcct aggtataatg ctagccccgg gaaaagatct aaagaggaga
       61 aagaattcga attcatgaaa accttgaagt tcaaactgta ccagcacaaa agaaatagat
      121 ttctcaaacg catgattaat gcaggtgggg caatctacaa ccattgtatt gctctacata
      181 aacgctacta tcgtatgtgg ggcaaacact taaactgtgc aaaacttcaa gctcatattg
      241 ccaaactgag aaagcgtaac ccattttggc aaacagtagg ttctcaggcg gtacaagata
      301 tttgtcaacg cattgagaaa gcctaccaat tattttttaa acacaacaag aagggggtta
      361 gaccaccagg atttaagaag gtaaaaaaat acaaatcatt tactctaaaa caagcaggtt
      421 ataagttttt ggatggaaat agagtaaaaa ttgggagtaa aatttatcag ttttggaagt
      481 ctagagaaat tgaggggaca gtcaaaacat taactattaa acgcacacca ttaggtgagt
      541 tgtttatggt tgtggtggtt gacgaaggta acaagccaga aaccaaattc atgactggta
      601 agatagcggg atttgatttc ggtttaaaaa cctttctcac ttgctcagac ggtacaacaa
      661 ttgagtctcc ccagtttttt aagcaatccc taaatgcaat taaaaaagcc agccgacagc
      721 attctaagaa attaaaaggt tcagccaatc gtgagcaagc tagaaagaac ttagtacgca
      781 ggtatgaaga tatctttaac cgtcgtcgtg attggttctg gaagttagcc catgagttaa
      841 cggataaatt tgatgttctc tgttttgaaa ccttaaacct taaaggaatg caacgacttt
      901 ggggaaggaa aatatcagac ttggcttttg gtgaattact gcaaattcta gaatgggttg
      961 ccaaaaacaa gaataaactg gttgttttca ttgacccgtg gtatcccagt agcaaaacct
     1021 gctctagctg tggacatatt ttagaaaacc tagatttgtc aattagacgc tggcgttgtc
     1081 cttcatgtca atctgaaaat gatcgggatg gaaacgctag tttgaatatt aaaagagtcg
     1141 ggagttcgac tctaggcgta ggagatgtta gacagtccca gactgcaatc tctgtcggat
     1201 cctgagagat ctttgacagc tagctcagtc ctaggtataa tactagtgaa aacctagatt
     1261 tgtcaattag acgctggcgt tgtccttcat gtcaatctga aaatgatcgg gatggaaacg
     1321 ctagtttgaa tattaaaaga gtcgggagtt cgactctagg cgtaggagat gttagacagt
     1381 cccagactgc aatctctgtc tgatgcctga atcccccgtt ttcaaaacgg gggagtggct
     1441 caagctcgga gatcatcatt gcgtttttta agcttggctg ttttggcgga tgagagaaga
     1501 ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc
     1561 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
     1621 gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa
     1681 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     1741 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     1801 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag
     1861 gccatcctga cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat
     1921 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1981 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2041 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2101 tcagttgggt gcagcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2161 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2221 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2281 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2341 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2401 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt acgcgccctg
     2461 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2521 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2581 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2641 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     2701 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2761 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt
     2821 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2881 taacaaaata ttaacgttta caatttaaaa ggatctaggt gaagatcctt tttgataatc
     2941 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3001 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3061 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3241 tgttaccagt caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc
     3301 ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgaccgg
     3361 gtcgaatttg ctttcgaatt tctgccattc atccgcttat tatcacttat tcaggcgtag
     3421 caaccaggcg tttaagggca ccaataactg ccttaaaaaa attacgcccc gccctgccac
     3481 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
     3541 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     3601 cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
     3661 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg
     3721 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
     3781 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
     3841 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
     3901 cggaactccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
     3961 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
     4021 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     4081 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     4141 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
     4201 ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc ccagggcttc
     4261 ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc cgtcacaggt
     4321 atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tactgattta gtgtatgatg
     4381 gtgtttttga ggtgctccag tggcttctgt ttctatcagc tgtccctcct gttcagctac
     4441 tgacggggtg gtgcgtaacg gcaaaagcac cgccggacat cagcgctagc ggagtgtata
     4501 ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa
     4561 aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc
     4621 tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg
     4681 gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc
     4741 aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa
     4801 tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc
     4861 cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc
     4921 gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg
     4981 actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc
     5041 ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat
     5101 ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt
     5161 ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc
     5221 ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga
     5281 ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta ggctcatgag
     5341 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     5401 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tctgctcatg
     5461 tttgacagct tatcatcgat
//
