LOCUS       sequence-162403-        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     complement(215..232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(325..913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(405..424)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1084..1875,1876..1944))
                     /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     1707..1726
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1945..2049)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2350..2372
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2364..2381
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2365..2381
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2392..2411
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2392..2410
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2475..2723
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2475..2495
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2646..2665
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        join(2742..2789,1..28)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta gagctagaaa tagcaagtta
       61 ggaccgagac ctgtacaacg cgtggtacct ggagcacaag actggcctca cgggccttcc
      121 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata
      181 gctgtttcct tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg
      241 tcgttcgggt aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg
      301 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
      361 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
      421 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
      481 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
      541 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
      601 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
      661 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
      721 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
      781 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
      841 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
      901 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
      961 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     1021 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     1081 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     1141 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     1201 ccccagtgct gcaatgatac cgcgagaacc acgctcaccg gctccagatt tatcagcaat
     1261 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     1321 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     1381 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     1441 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     1501 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     1561 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     1621 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     1681 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     1741 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     1801 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     1861 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     1921 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     1981 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2041 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     2101 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     2161 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtggccg ctacagggcg
     2221 ctcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgtttcggtg cgggcctctt
     2281 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     2341 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgac gtaatacgac
     2401 tcactatagg gcgaattggc ggaaggccgt caaggccacg tgtcttgtcc agagctcgga
     2461 tccggtctcc catggagggc ctatttccca tgattccttc atatttgcat atacgataca
     2521 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     2581 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaag ttatgtttta
     2641 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     2701 tcttgtggaa aggacgaaac accgggtctt cgagaagacc tgttttagag ctagaaatag
     2761 caagttaaaa taaggctagt ccgttatca
//
