LOCUS       sequence-449951-        2280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human gRNA expression vector backbone with BsmBI Golden Gate site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2280)
  AUTHORS   Osgood NRB, Zawalick NM, Sawyer CB, Cowan QT, Gu S, Mawson SJ, 
            Ranzau BL, Li L, Gymrek M, Goren A, Komor AC
  TITLE     Genome editing with programmable base editors in human cells
  JOURNAL   Methods in Enzymology, February 5, 2025
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: "Methods in 
            Enzymology, February 5, 2025"
COMMENT     Sequence Label: pU6-GG-gRNA
FEATURES             Location/Qualifiers
     source          1..2280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(541..1332,1333..1401))
                     /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     1164..1183
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1402..1506)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1613..1861
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1613..1633
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1784..1803
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1883..1958
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(join(2062..2280,1..370))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2142..2161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
       61 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
      121 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
      181 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
      241 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
      301 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
      361 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
      421 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
      481 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
      541 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
      601 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      661 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      721 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      781 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      841 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      901 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      961 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     1021 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     1081 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     1141 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     1201 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     1261 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     1321 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     1381 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     1441 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     1501 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgct agctgtacaa aaaagcaggc
     1561 tttaaaggaa ccaattcagt cgactggatc cggtaccaag gtcgggcagg aagagggcct
     1621 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
     1681 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     1741 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     1801 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     1861 cagagacgca ttaatcgtct cagtttaaga gctagaaata gcaagtttaa ataaggctag
     1921 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttaagct tgggccgctc
     1981 gaggtacctc tctacatatg acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     2041 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2101 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2161 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2221 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
//
