LOCUS       sequence-416230-        2204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for human U6 promoter driven SpCas9-based pegRNAs, 
            comprised of hU6-[BsmBI]-terminator (spacer and RTT/PBS oligos must 
            be cloned in).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2204)
  AUTHORS   Ferreira da Silva J, Tou CJ, King EM, Eller ML, Rufino-Ramos D, Ma 
            L, Cromwell CR, Metovic J, Benning FMC, Chao LH, Eichler FS, 
            Kleinstiver BP
  TITLE     Click editing enables programmable genome writing using DNA 
            polymerases and HUH endonucleases.
  JOURNAL   Nat Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x.
  PUBMED    39039307
REFERENCE   2  (bases 1 to 2204)
  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 
            Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x."
COMMENT     Sequence Label: pegRNA entry vector - pUC19-U6-[BsmBI_entry]-t...
FEATURES             Location/Qualifiers
     source          1..2204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     82..102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     253..272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(455..1043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(535..554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1214..2005,2006..2074))
                     /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     1837..1856
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2075..2179)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc ggagacgatt aatgcgtctc ctttttttaa
      361 gcttgggccg ctcgaggtac ctctctacat atgacatgtg agcaaaaggc cagcaaaagg
      421 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
      481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
      541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
      601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
      661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
      721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
      781 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
      841 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
      901 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
      961 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     1021 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     1081 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     1141 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     1201 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     1261 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     1321 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     1381 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1441 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     1501 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     1561 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     1621 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     1681 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     1741 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     1801 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     1861 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     1921 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     1981 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     2041 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     2101 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2161 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acct
//
