LOCUS       sequence-162407-        2789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-based cloning of SpCas9 gRNA guide sequence, position 4/14/24 
            in the array.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2789)
  AUTHORS   Vad-Nielsen J, Lin L, Bolund L, Nielsen AL, Luo Y
  TITLE     Golden Gate Assembly of CRISPR gRNA expression array for 
            simultaneously targeting multiple genes.
  JOURNAL   Cell Mol Life Sci. 2016 May 13.
  PUBMED    27178736
REFERENCE   2  (bases 1 to 2789)
  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: "Cell Mol 
            Life Sci. 2016 May 13."
COMMENT     Sequence Label: pMA-SpCas9-g4
FEATURES             Location/Qualifiers
     source          1..2789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(607..1398,1399..1467))
                     /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     1230..1249
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1468..1572)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1873..1895
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1887..1904
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1888..1904
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1915..1934
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1915..1933
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1998..2246
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1998..2018
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2169..2188
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2265..2340
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2527..2544)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2637..2789,1..436))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2717..2736)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
       61 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
      121 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
      181 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
      241 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
      301 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
      361 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
      421 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
      481 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
      541 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
      601 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
      661 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
      721 tggccccagt gctgcaatga taccgcgaga accacgctca ccggctccag atttatcagc
      781 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
      841 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
      901 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
      961 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     1021 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     1081 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     1141 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     1201 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     1261 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     1321 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     1381 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     1441 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     1501 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     1561 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     1621 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     1681 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgg ccgctacagg
     1741 gcgctcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgtttcg gtgcgggcct
     1801 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     1861 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gacgtaatac
     1921 gactcactat agggcgaatt ggcggaaggc cgtcaaggcc acgtgtcttg tccagagctc
     1981 ggatccggtc tcgccaggag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2041 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     2101 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2161 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2221 atatcttgtg gaaaggacga aacaccgggt cttcgagaag acctgtttta gagctagaaa
     2281 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2341 ttttttgttt tagagctaga aatagcaagt tatgttggag acctgtacaa cgcgtggtac
     2401 ctggagcaca agactggcct cacgggcctt ccgctcactg cccgctttcc agtcgggaaa
     2461 cctgtcgtgc cagctgcatt aacatggtca tagctgtttc cttgcgtatt gggcgctctc
     2521 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg gtaaagcctg gggtgcctaa
     2581 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2641 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2701 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2761 cctgttccga ccctgccgct taccggata
//
