LOCUS       sequence-354254-        2280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transiently expressing a nicking sgRNA to facilitate the 
            introduction of HEK3 CTT insertion in PE3 approach.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2280)
  AUTHORS   Li H, Busquets O, Verma Y, Syed KM, Kutnowski N, Pangilinan GR, 
            Gilbert LA, Bateup HS, Rio DC, Hockemeyer D, Soldner F
  TITLE     Highly efficient generation of isogenic pluripotent stem cell models
            using prime editing.
  JOURNAL   Elife. 2022 Sep 7;11:e79208. doi: 10.7554/eLife.79208.
  PUBMED    36069759
REFERENCE   2  (bases 1 to 2280)
  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: "Elife."; 
            date: "2022-09-7"; volume: "11:e79208. doi"; pages: " 
            10.7554/eLife.79208"
COMMENT     Sequence Label: pBPK1520-HEK3-CTTins-ng
FEATURES             Location/Qualifiers
     source          1..2280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(83..671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(163..182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(842..1633,1634..1702))
                     /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     1465..1484
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1703..1807)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1914..2162
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1914..1934
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2085..2104
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2184..2259
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cgaggtacct ctctacatat gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
       61 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      121 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      181 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      241 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
      301 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
      361 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
      421 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
      481 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
      541 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
      601 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
      661 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
      721 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
      781 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
      841 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
      901 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
      961 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     1021 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     1081 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     1141 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     1201 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     1261 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     1321 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     1381 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     1441 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     1501 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     1561 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     1621 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     1681 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     1741 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     1801 ttccgcgcac atttccccga aaagtgccac ctgacgtcgc tagctgtaca aaaaagcagg
     1861 ctttaaagga accaattcag tcgactggat ccggtaccaa ggtcgggcag gaagagggcc
     1921 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
     1981 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     2041 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     2101 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     2161 ccggtcaacc agtatcccgg tgcgttttag agctagaaat agcaagttaa aataaggcta
     2221 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttaagc ttgggccgct
//
