LOCUS       sequence-159020-        2789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-based cloning of SpCas9 gRNA guide sequence, position 2/12/22 
            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-g2
FEATURES             Location/Qualifiers
     source          1..2789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     278..300
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     292..309
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        403..651
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     403..423
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     574..593
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        670..745
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(932..949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1042..1630)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1122..1141)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1801..2592,2593..2661))
                     /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     2424..2443
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2662..2766)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca
      361 aggccacgtg tcttgtccag agctcggatc cggtctccca tggagggcct atttcccatg
      421 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
      481 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
      541 tagtttgcag ttttaaagtt atgttttaaa atggactatc atatgcttac cgtaacttga
      601 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgggtcttcg
      661 agaagacctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      721 tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttagga
      781 ccgagacctg tacaacgcgt ggtacctgga gcacaagact ggcctcacgg gccttccgct
      841 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaacat ggtcatagct
      901 gtttccttgc gtattgggcg ctctccgctt cctcgctcac tgactcgctg cgctcggtcg
      961 ttcgggtaaa gcctggggtg cctaatgagc aaaaggccag caaaaggcca ggaaccgtaa
     1021 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1081 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1141 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1201 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1261 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1321 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1381 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1441 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     1501 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1561 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1621 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1681 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     1741 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     1801 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     1861 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     1921 cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa
     1981 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2041 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2101 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2161 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2221 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2281 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     2341 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     2401 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     2461 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2521 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     2581 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     2641 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2701 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     2761 tccgcgcaca tttccccgaa aagtgccac
//
