LOCUS       sequence-342966-        2564 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Delivery of guide RNA for prime editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2564)
  AUTHORS   Tong Y, Jorgensen TS, Whitford CM, Weber T, Lee SY
  TITLE     A versatile genetic engineering toolkit for E. coli based on 
            CRISPR-prime editing.
  JOURNAL   Nat Commun. 2021 Sep 1;12(1):5206. doi: 10.1038/s41467-021-25541-3.
  PUBMED    34471126
REFERENCE   2  (bases 1 to 2564)
  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: "2021-09-1"; volume: "12(1):5206. doi"; pages: " 
            10.1038/s41467-021-25541-3"
COMMENT     Sequence Label: pPEgRNA
FEATURES             Location/Qualifiers
     source          1..2564
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..71
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        72..147
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             168..197
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             213..230
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(286..303)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      456..502
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(555..572)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(726..1314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(806..825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1485..2276,2277..2345))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2108..2127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2346..2450)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2518..2536
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(join(2540..2564,1..19))
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 tttcgctaag gatgatttct ggaattctaa agatctttga cagctagctc agtcctaggt
       61 ataatactag tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      121 cttgaaaaag tggcaccgag tcggtgcttt ttttgaagct tgggcccgaa caaaaactca
      181 tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttaaa
      241 cggtctccag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa
      301 atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt
      361 cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg
      421 gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga
      481 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa ctggatcctt actcgagtct
      541 agactgcagg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
      601 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
      661 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      721 gcgtttttcc acaggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      781 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      841 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      901 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      961 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1021 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1081 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1141 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     1201 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1261 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1321 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1381 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1441 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1501 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     1561 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1621 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1681 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1741 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1801 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1861 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1921 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1981 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2041 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2101 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2161 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     2221 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2281 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2341 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2401 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2461 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     2521 aggcgtatca cgaggcagaa tttcagataa aaaaaatcct tagc
//
