LOCUS       sequence-162402-        2789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-based cloning of SpCas9 gRNA guide sequence, position 1/11/21 
            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-g1
FEATURES             Location/Qualifiers
     source          1..2789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        42..290
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     42..62
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     213..232
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        309..384
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(571..588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(681..1269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(761..780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1440..2231,2232..2300))
                     /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     2063..2082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2301..2405)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2706..2728
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2720..2737
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2721..2737
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2748..2767
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2748..2766
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggccacgtgt cttgtccaga gctcggatcc ggtctcgcta tgagggccta tttcccatga
       61 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
      121 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      181 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      241 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gggtcttcga
      301 gaagacctgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
      361 gaaaaagtgg caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttacatg
      421 ggagacctgt acaacgcgtg gtacctggag cacaagactg gcctcacggg ccttccgctc
      481 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaacatg gtcatagctg
      541 tttccttgcg tattgggcgc tctccgcttc ctcgctcact gactcgctgc gctcggtcgt
      601 tcgggtaaag cctggggtgc ctaatgagca aaaggccagc aaaaggccag gaaccgtaaa
      661 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      721 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      781 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      841 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
      901 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      961 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1021 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1081 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     1141 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1201 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1261 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1321 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1381 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     1441 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1501 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1561 agtgctgcaa tgataccgcg agaaccacgc tcaccggctc cagatttatc agcaataaac
     1621 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     1681 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     1741 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     1801 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     1861 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     1921 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     1981 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     2041 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     2101 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     2161 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     2221 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2281 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2341 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2401 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     2461 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     2521 tcccttataa atcaaaagaa tagaccgaga tagggttgag tggccgctac agggcgctcc
     2581 cattcgccat tcaggctgcg caactgttgg gaagggcgtt tcggtgcggg cctcttcgct
     2641 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     2701 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgacgtaa tacgactcac
     2761 tatagggcga attggcggaa ggccgtcaa
//
