LOCUS       sequence-338872-        2476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  single guide RNA (sgRNA) and Prime editing guide RNA (pegRNA) 
            cloning and mammalian cell expression. BbsI cloning for sgRNAs and 
            BbsI/PstI for pegRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2476)
  AUTHORS   Standage-Beier K, Tekel SJ, Brafman DA, Wang X
  TITLE     Prime Editing Guide RNA Design Automation Using PINE-CONE.
  JOURNAL   ACS Synth Biol. 2021 Feb 19;10(2):422-427. doi: 
            10.1021/acssynbio.0c00445. Epub 2021 Jan 19.
  PUBMED    33464043
REFERENCE   2  (bases 1 to 2476)
  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"; doi: 
            "10.1021/acssynbio.0c00445"; journalName: "ACS Synth Biol"; date: 
            "2021-02-19- 19"; volume: "10"; issue: "2"; pages: "422-427"
COMMENT     Sequence Label: pHSG1C3
FEATURES             Location/Qualifiers
     source          1..2476
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        274..349
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      450..507
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(531..548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(702..1290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(782..801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1473..1567)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1588..2247)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2162..2181
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2409..2427
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2431..2474)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattcgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccgggtc ttcgagaaga cctgttttag agctagaaat agcaagttaa
      301 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt
      361 agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct
      421 gcagacaaat ctagtagcgg ccgctgcagt ccggcaaaaa agggcaaggt gtcaccaccc
      481 tgcccttttt ctttaaaacc gaaaagatta cttcgcgtta tgcaggcttc ctcgctcact
      541 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      601 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      661 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccacag gctccgcccc
      721 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      781 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      841 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      901 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      961 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1021 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1081 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1141 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1201 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1261 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1321 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1381 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     1441 gagtaaactt ggtctgacag ctcgaggctt ggattctcac caataaaaaa cgcccggcgg
     1501 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     1561 aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg
     1621 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     1681 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     1741 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     1801 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     1861 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     1921 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     1981 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg
     2041 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     2101 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     2161 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     2221 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
     2281 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac
     2341 gtgcccgatc aactcgagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     2401 cctataaaaa taggcgtatc acgaggcaga atttcagata aaaaaaatcc ttagctttcg
     2461 ctaaggatga tttctg
//
