LOCUS       sequence-342967-        3891 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 3891)
  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 3891)
  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: pnsgRNA
FEATURES             Location/Qualifiers
     source          1..3891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..34
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        54..88
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        89..164
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             185..214
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             230..247
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(303..320)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      473..559
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1097..2047)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2095..2317
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(2809..2903)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2934..3728)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3045..3064)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3655..3674
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 acattaacct ataaaaatag gcgtatcacg aggcccttga attctaaaga tctttgacag
       61 ctagctcagt cctaggtata atactagtgt tttagagcta gaaatagcaa gttaaaataa
      121 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaagcttgg
      181 gcccgaacaa aaactcatct cagaagagga tctgaatagc gccgtcgacc atcatcatca
      241 tcatcattga gtttaaacgg tctccagctt ggctgttttg gcggatgaga gaagattttc
      301 agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc
      361 ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc
      421 gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa
      481 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
      541 tctcctgagt aggacaaatc cgccgcccta gacctagggt acgggttttg ctgcccgcaa
      601 acgggctgtt ctggtgttgc tagtttgtta tcagaatcgc agatccggct tcagccggtt
      661 tgccggctga aagcgctatt tcttccagaa ttgccatgat tttttcccca cgggaggcgt
      721 cactggctcc cgtgttgtcg gcagctttga ttcgataagc agcatcgcct gtttcaggct
      781 gtctatgtgt gactgttgag ctgtaacaag ttgtctcagg tgttcaattt catgttctag
      841 ttgctttgtt ttactggttt cacctgttct attaggtgtt acatgctgtt catctgttac
      901 attgtcgatc tgttcatggt gaacagcttt gaatgcacca aaaactcgta aaagctctga
      961 tgtatctatc ttttttacac cgttttcatc tgtgcatatg gacagttttc cctttgatat
     1021 gtaacggtga acagttgttc tacttttgtt tgttagtctt gatgcttcac tgatagatac
     1081 aagagccata agaacctcag atccttccgt atttagccag tatgttctct agtgtggttc
     1141 gttgtttttg cgtgagccat gagaacgaac cattgagatc atacttactt tgcatgtcac
     1201 tcaaaaattt tgcctcaaaa ctggtgagct gaatttttgc agttaaagca tcgtgtagtg
     1261 tttttcttag tccgttatgt aggtaggaat ctgatgtaat ggttgttggt attttgtcac
     1321 cattcatttt tatctggttg ttctcaagtt cggttacgag atccatttgt ctatctagtt
     1381 caacttggaa aatcaacgta tcagtcgggc ggcctcgctt atcaaccacc aatttcatat
     1441 tgctgtaagt gtttaaatct ttacttattg gtttcaaaac ccattggtta agccttttaa
     1501 actcatggta gttattttca agcattaaca tgaacttaaa ttcatcaagg ctaatctcta
     1561 tatttgcctt gtgagttttc ttttgtgtta gttcttttaa taaccactca taaatcctca
     1621 tagagtattt gttttcaaaa gacttaacat gttccagatt atattttatg aattttttta
     1681 actggaaaag ataaggcaat atctcttcac taaaaactaa ttctaatttt tcgcttgaga
     1741 acttggcata gtttgtccac tggaaaatct caaagccttt aaccaaagga ttcctgattt
     1801 ccacagttct cgtcatcagc tctctggttg ctttagctaa tacaccataa gcattttccc
     1861 tactgatgtt catcatctga gcgtattggt tataagtgaa cgataccgtc cgttctttcc
     1921 ttgtagggtt ttcaatcgtg gggttgagta gtgccacaca gcataaaatt agcttggttt
     1981 catgctccgt taagtcatag cgactaatcg ctagttcatt tgctttgaaa acaactaatt
     2041 cagacataca tctcaattgg tctaggtgat tttaatcact ataccaattg agatgggcta
     2101 gtcaatgata attactagtc cttttccttt gagttgtggg tatctgtaaa ttctgctaga
     2161 cctttgctgg aaaacttgta aattctgcta gaccctctgt aaattccgct agacctttgt
     2221 gtgttttttt tgtttatatt caagtggtta taatttatag aataaagaaa gaataaaaaa
     2281 agataaaaag aatagatccc agccctgtgt ataactcact actttagtca gttccgcagt
     2341 attacaaaag gatgtcgcaa acgctgtttg ctcctctaca aaacagacct taaaacccta
     2401 aaggcttaag tagcaccctc gcaagctcgg gcaaatcgct gaatattcct tttgtctccg
     2461 accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc gctcacggct
     2521 ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca tgcaaggaaa
     2581 ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat ggcgggtctg
     2641 ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga ttttccagtc
     2701 tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac ccagtaaggc
     2761 agcggtatca tcaacaggct tacccgtctt actgtcccta gtgcttggat tctcaccaat
     2821 aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc agatggagtt ctgaggtcat
     2881 tactggatct atcaacagga gtccaagcga gctctcgaac cccagagtcc cgctcagaag
     2941 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     3001 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     3061 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     3121 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
     3181 tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     3241 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
     3301 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     3361 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     3421 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     3481 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     3541 tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     3601 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     3661 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     3721 tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag atcttgatcc cctgcgccat
     3781 cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca
     3841 gagggcgccc cagctggcaa ttccgacgtc taagaaacca ttattatcat g
//
