LOCUS       sequence-162408-        2789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-based cloning of SpCas9 gRNA guide sequence, position 5/15/25 
            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-g5
FEATURES             Location/Qualifiers
     source          1..2789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..56
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_RNA        complement(243..318)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(337..585)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(395..414)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(565..585)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(649..668)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(650..668)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(679..696)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(679..695)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(688..710)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        1011..1115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1116..1184,1185..1976)
                     /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     complement(1334..1353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2147..2735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2636..2655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattaggcac cccaggcttt acccgaacga ccgagcgcag cgagtcagtg agcgaggaag
       61 cggagagcgc ccaatacgca aggaaacagc tatgaccatg ttaatgcagc tggcacgaca
      121 ggtttcccga ctggaaagcg ggcagtgagc ggaaggcccg tgaggccagt cttgtgctcc
      181 aggtaccacg cgttgtacag gtctcctgca taacttgcta tttctagctc taaaacaaaa
      241 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
      301 tatttctagc tctaaaacag gtcttctcga agacccggtg tttcgtcctt tccacaagat
      361 atataaagcc aagaaatcga aatactttca agttacggta agcatatgat agtccatttt
      421 aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt
      481 ttgtactaat atctttgtgt ttacagtcaa attaattcca attatctctc taacagcctt
      541 gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcaacag gagaccggat
      601 ccgagctctg gacaagacac gtggccttga cggccttccg ccaattcgcc ctatagtgag
      661 tcgtattacg tcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
      721 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
      781 agaggcccgc accgaaacgc ccttcccaac agttgcgcag cctgaatggc gaatgggagc
      841 gccctgtagc ggccactcaa ccctatctcg gtctattctt ttgatttata agggattttg
      901 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
      961 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1021 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1081 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1141 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1201 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1261 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1321 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     1381 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1441 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1501 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1561 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1621 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     1681 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1741 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1801 ttgctgataa atctggagcc ggtgagcgtg gttctcgcgg tatcattgca gcactggggc
     1861 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1921 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1981 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2041 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2101 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2161 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2221 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2281 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2341 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2401 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2461 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2521 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2581 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2641 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2701 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2761 ggttcctggc cttttgctgg ccttttgct
//
