LOCUS       sequence-440881-        2301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden gate backbone for mammalian expression of Sp-gRNA with 
            3'-MS2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2301)
  AUTHORS   Cowan QT, Gu S, Gu W, Ranzau BL, Simonson TS, Komor AC
  TITLE     Development of multiplexed orthogonal base editor (MOBE) systems.
  JOURNAL   Nat Biotechnol. 2024 May 21. doi: 10.1038/s41587-024-02240-0.
  PUBMED    38773305
REFERENCE   2  (bases 1 to 2301)
  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 May 21. doi: 10.1038/s41587-024-02240-0."
COMMENT     Sequence Label: 3'MS2-gRNA GG backbone (MOBE1-2)
FEATURES             Location/Qualifiers
     source          1..2301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        142..390
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     142..162
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     313..332
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        408..483
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        488..506
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      complement(612..1200)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(692..711)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1371..2162,2163..2231))
                     /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     1994..2013
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(2232..2301,1..35))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
       61 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
      121 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      181 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      241 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      301 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      361 ttatatatct tgtggaaagg acgaaacacc ggggtcttcg agaagacgtt ttagagctag
      421 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
      481 tgcgcgcaca tgaggatcac ccatgtgctt tttttaagct tgggccgctc gaggtacctc
      541 tctacatatg acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      601 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      661 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      721 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
      781 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
      841 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
      901 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
      961 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1021 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1081 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1141 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1201 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1261 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1321 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     1381 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     1441 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     1501 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     1561 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     1621 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     1681 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     1741 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     1801 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     1861 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     1921 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     1981 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     2041 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2101 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2161 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2221 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     2281 atgagcggat acatatttga a
//
