LOCUS       sequence-296571-        2363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a pegRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2363)
  AUTHORS   Schmid-Burgk JL, Gao L, Li D, Gardner Z, Strecker J, Lash B, Zhang F
  TITLE     Highly Parallel Profiling of Cas9 Variant Specificity.
  JOURNAL   Mol Cell. 2020 Mar 17. pii: S1097-2765(20)30143-X. doi: 
            10.1016/j.molcel.2020.02.023.
  PUBMED    32187529
REFERENCE   2  (bases 1 to 2363)
  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: "Mol Cell. 
            2020 Mar 17. pii: S1097-2765(20)30143-X. doi: 
            10.1016/j.molcel.2020.02.023."
COMMENT     Sequence Label: pU6-pegRNA-EMX1+38
FEATURES             Location/Qualifiers
     source          1..2363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(9..113)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        220..468
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     220..240
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     391..410
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        490..565
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    596..633
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     rep_origin      complement(752..1340)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(832..851)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1511..2302,2303..2363,1..8))
                     /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     2134..2153
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
       61 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      121 ccccgaaaag tgccacctga cgtcgctagc tgtacaaaaa agcaggcttt aaaggaacca
      181 attcagtcga ctggatccgg taccaaggtc gggcaggaag agggcctatt tcccatgatt
      241 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
      301 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      361 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      421 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt gctccagagg
      481 ccccccttgg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      541 tgaaaaagtg gcaccgagtc ggtgctgtag cctgccctct gcacctcctc accaaggctt
      601 gtcgacgacg gcggtctccg tcgtcaggat catggggggc ctctggagtt tttttaagct
      661 tgggccgctc gaggtacctc tctacatatg acatgtgagc aaaaggccag caaaaggcca
      721 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
      781 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
      841 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
      901 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
      961 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1021 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1081 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     1141 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     1201 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     1261 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     1321 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     1381 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     1441 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     1501 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     1561 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     1621 catctggccc cagtgctgca atgataccgc gagatccacg ctcaccggct ccagatttat
     1681 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     1741 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     1801 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     1861 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     1921 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     1981 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     2041 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     2101 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     2161 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     2221 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     2281 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     2341 taagggcgac acggaaatgt tga
//
