LOCUS       sequence-423076-        5890 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the pegRNA for genome editing in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5890)
  AUTHORS   Zhang H, Ma J, Wu Z, Chen X, Qian Y, Chen W, Wang Z, Zhang Y, Zhu H,
            Huang X, Ji Q
  TITLE     BacPE: a versatile prime-editing platform in bacteria by inhibiting 
            DNA exonucleases.
  JOURNAL   Nat Commun. 2024 Jan 27;15(1):825. doi: 10.1038/s41467-024-45114-4.
  PUBMED    38280845
REFERENCE   2  (bases 1 to 5890)
  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 
            Commun."; date: "2024-01-27"; volume: "15(1):825. doi"; pages: " 
            10.1038/s41467-024-45114-4"
COMMENT     Sequence Label: pegRNA2_tevopreQ1
FEATURES             Location/Qualifiers
     source          1..5890
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        181..215
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     ncRNA           274..341
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      352..399
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(474..491)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(645..1233)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(725..744)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1404..2219)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1404..2219)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2127..2146
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2220..2324)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2392..2410
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     4105..4127
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4119..4136
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4120..4136
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4173..4189
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4174..4190
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(4252..5586,5587..5673))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
ORIGIN
        1 tctcgtttgg attgcaactg gtctattttc ctcttttgtt tgatagaaaa tcataaaagg
       61 atttgcagac tacgggccta aagaactaaa aaatctatct gtttcttttc attctctgta
      121 ttttttatag tttctgttgc atgggcataa agttgcaagc ttgatatcac gctgtggaag
      181 ttgacagcta gctcagtcct aggtataata ctagtcgaga ccatcgctag gtctcacgcg
      241 gttctatcta gttacgcgtt aaaccaacta gaaggccggc atggtcccag cctcctcgct
      301 ggcgccggct gggcaacatg cttcggcatg gcgaatggga cacgtatcat gctagcataa
      361 ccccttgggg cctctaaacg ggtcttgagg ggttttttgg gtatcgaggt gcattaatga
      421 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
      481 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
      541 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
      601 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
      661 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
      721 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
      781 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
      841 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
      901 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
      961 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     1021 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     1081 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     1141 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     1201 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     1261 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     1321 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     1381 tatgagtaaa cttggtctga cagttagaaa aactcatcga gcatcaaatg aaactgcaat
     1441 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1501 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     1561 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     1621 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     1681 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     1741 aaaccgttat tcattcgtga ttgcgcctga gcgagtcgaa atacgcgatc gctgttaaaa
     1801 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     1861 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccagggatc
     1921 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     1981 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     2041 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     2101 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     2161 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     2221 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     2281 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     2341 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     2401 atcacgaggc cctttttgac agctagctca gtccgagacg atcgctacgt ctcatcacag
     2461 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2521 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     2581 ataggatttt aacattttgc gttgttccaa aagttatcaa cagcctagaa cgtcatagga
     2641 agcgattaca gacactttat agctatcagc atgggaacat aagggcagga tgaaatatgg
     2701 gtcttaaaac gcaaatggtg aggttttaga ggtatttttt gaagatgatt aaggcggttt
     2761 gtttttaaaa tttttggcgg ctctcaggct gcttacattt taaccagttc agtgaaaagt
     2821 tctttttcag caaatttctg tttagcacca tagctaaaac ttgcgtggaa catattaaga
     2881 attgaccgaa atgacacaat ctcaattata ttttttttga aaagttttct ttatcaaata
     2941 ttttaaatca ttgatttata tataagtatg cattcatttt aataattaat ctttatttaa
     3001 caatgattta tctatattca attgtttaat tattcttact aatattatct ctatatcaat
     3061 attttttatt taaaaacata tgtttagtag tgcttttgat taaagtacca gagggaggga
     3121 gcagagctga atgggaaata ctcaccctag agcgattctt aaaaatcacc ctaaagtatt
     3181 cccattcgat gtaccgtcgg tcggtcgctt tcgcatcagg gatgacatca ctgtatcaag
     3241 ctgccactgt tatgattacg attgatagca ccgcctgaac acgctcataa ccgccaatta
     3301 aatgactact cattgcgtcc gctactctgt tcagttcccc tagtaatagc gtttttccga
     3361 tgtgtgctag cgtcactgta cctcatcacc cacacatgga cagattgagt tacagacatt
     3421 ggctaaattt ttggggtcta tgcttgacaa agcgagttaa aaccttagaa tttaagacag
     3481 gtacattaag cccctgtggt gaaatcatta gcggtcgtca aaccttaatg ctttcgattg
     3541 ccgatatgtc agtatcgaag atctgtaccc cataaattac ggtaaagccc caagcaattg
     3601 caaggggctt ttatcttttt taacaaaaaa aatttataaa tcaggatttt ataacactaa
     3661 ataatccaaa gtacatgagt aagtcatgac cactcctgcg atgtgtgtag cactctgagt
     3721 atccgtatta tatcagtgat gtcatataca accacataat gcggatgtat cactaactcc
     3781 ctagtatctt tctgtctgcc tgtgcgcccc atcagtcgat tatcaacaag taaatctgtc
     3841 ttttcttcaa ttaaatcatc aatttcaata gctgctatag ggttgcgttc ttcaagataa
     3901 tcatagatat ttctacgatc ttggctatgc ggtgtgaaat accgcacaga tgcgtaagga
     3961 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     4021 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     4081 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
     4141 tgacgcgtat tgggatggta ccgggccccc cctcgaggtc gacggtatcg atacggcatc
     4201 agagcagatt gtactgagag tgcaccataa tcggcatttt cttttgcgtt tttatttgtt
     4261 aactgttaat tgtccttgtt caaggatgct gtctttgaca acagatgttt tcttgccttt
     4321 gatgttcagc aggaagctag gcgcaaacgt tgattgtttg tctgcgtaga atcctctgtt
     4381 tgtcatatag cttgtaatca cgacattgtt tcctttcgct tgaggtacag cgaagtgtga
     4441 gtaagtaaag gttacatcgt taggatcaag atccattttt aacacaaggc cagttttgtt
     4501 cagcggcttg tatgggccag ttaaagaatt agaaacataa ccaagcatgt aaatatcgtt
     4561 agacgtaatg ccgtcaatcg tcatttttga tccgcgggag tcagtgaaca gataccattt
     4621 gccgttcatt ttaaagacgt tcgcgcgttc aatttcatct gttactgtgt tagatgcaat
     4681 cagcggtttc atcacttttt tcagtgtgta atcatcgttt agctcaatca taccgagagc
     4741 gccgtttgct aactcagccg tgcgtttttt atcgctttgc agaagttttt gactttcttg
     4801 acggaagaat gatgtgcttt tgccatagta tgctttgtta aataaagatt cttcgccttg
     4861 gtagccatct tcagttccag tgtttgcttc aaatactaag tatttgtggc ctttatcttc
     4921 tacgtagtga ggatctctca gcgtatggtt gtcgcctgag ctgtagttgc cttcatcgat
     4981 gaactgctgt acattttgat acgtttttcc gtcaccgtca aagattgatt tataatcctc
     5041 tacaccgttg atgttcaaag agctgtctga tgctgatacg ttaacttgtg cagttgtcag
     5101 tgtttgtttg ccgtaatgtt taccggagaa atcagtgtag aataaacgga tttttccgtc
     5161 agatgtaaat gtggctgaac ctgaccattc ttgtgtttgg tcttttagga tagaatcatt
     5221 tgcatcgaat ttgtcgctgt ctttaaagac gcggccagcg tttttccagc tgtcaataga
     5281 agtttcgccg actttttgat agaacatgta aatcgatgtg tcatccgcat ttttaggatc
     5341 tccggctaat gcaaagacga tgtggtagcc gtgatagttt gcgacagtgc cgtcagcgtt
     5401 ttgtaatggc cagctgtccc aaacgtccag gccttttgca gaagagatat ttttaattgt
     5461 ggacgaatcg aactcaggaa cttgatattt ttcatttttt tgctgttcag ggatttgcag
     5521 catatcatgg cgtgtaatat gggaaatgcc gtatgtttcc ttatatggct tttggttcgt
     5581 ttctttcgca aacgcttgag ttgcgcctcc tgccagcagt gcggtagtaa aggttaatac
     5641 tgttgcttgt tttgcaaact ttttgatgtt catcgttcat gtctcctttt ttatgtactg
     5701 tgttagcggt ctgcttcttc cagccctcct gtttgaagat ggcaagttag ttacgcacaa
     5761 taaaaaaaga cctaaaatat gtaaggggtg acgccaaagt atacactttg ccctttacac
     5821 attttaggtc ttgcctgctt tatcagtaac aaacccgcgc gatttacttt tcgacctcat
     5881 tctattagac
//
