LOCUS       sequence-440939-        2296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden gate backbone for mammalian expression of Sp-gRNA with 
            SL3-MS2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2296)
  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 2296)
  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: SL3-MS2-gRNA GG backbone (MOBE3-4)
FEATURES             Location/Qualifiers
     source          1..2296
                     /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        477..495
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      complement(607..1195)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(687..706)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1366..2157,2158..2226))
                     /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     1989..2008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(2227..2296,1..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 accggcacat
      481 gaggatcacc catgtgccgg tgcttttttt aagcttgggc cgctcgaggt acctctctac
      541 atatgacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      601 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      661 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      721 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
      781 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      841 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      901 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
      961 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     1021 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     1081 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     1141 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     1201 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     1261 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     1321 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     1381 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1441 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1501 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1561 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     1621 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     1681 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     1741 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     1801 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     1861 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     1921 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     1981 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     2041 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     2101 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     2161 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     2221 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     2281 cggatacata tttgaa
//
