LOCUS       sequence-423155-        3207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for Cloning of multiple gRNAs driven by distinct promoters 
            (tRNA-Gln and synthetic U6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3207)
  TITLE     Multiple gRNA Cloning Vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3207)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pUC-tRNAGln-BbsI-sU6-Kan
FEATURES             Location/Qualifiers
     source          1..3207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     703..722
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(796..900)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     968..986
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1024..1046)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1146..1165
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1359..1381
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1373..1390
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1374..1390
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        1620..1695
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     1702..1722
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2026..2042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2026..2042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2039..2061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2050..2066
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2074..2080,2081..2098,2099..2104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2119..2140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2257..2274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2428..3016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2508..2527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3187..3207,1..795))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRFAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
       61 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
      121 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
      181 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
      241 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
      301 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga aacgaaatac
      361 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
      421 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
      481 tgttttccca gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
      541 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
      601 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
      661 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
      721 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
      781 ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc agggttattg
      841 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      901 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
      961 ctataaaaat aggcgtatca cgaggccctt ttgtctcgcg cgtttcggtg atgacggtga
     1021 aaacctctga cacatgcagc tcccggagac tgtcacagct tgtctgtaag cggatgccgg
     1081 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     1141 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     1201 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     1261 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     1321 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     1381 gacggccagt gaattgacgc gtattgggat accggttcta gatacgtctc tgtttaagag
     1441 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     1501 tggcaccgag tcggtgcttt tttggttcca tggtgtaatg gttagcactc tggactctga
     1561 atccagcgat ccgagttcaa atctcggtgg aacctgtcac cgggtcttcg agaagacctg
     1621 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     1681 gcaccgagtc ggtgcttttt tgagggccta tttcccatga ttccttcata tttgcatata
     1741 atatttccta gtaactatag aggcttaatg tgcgataaaa gacagataat ctgttctttt
     1801 taatactagc tacattttac atgataggct tggatttcta taagagatac aaatactaaa
     1861 ttattatttt aaaaaacagc acaaaaggaa acttaccgta acttgaaagt aattgtgtgt
     1921 tttgagactt tatatatgca tgcgagaaaa gccttgtttg gtttggagac ggtgaattcg
     1981 gtaccgctag catcccaatg gcgcgccgag cttggctcga gcatggtcat agctgtttcc
     2041 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2101 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2161 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2221 gagaggcggt ttgcgtattg ggcgctgttc cgcttcctcg ctcactgact cgctgcgctc
     2281 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2341 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2401 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2461 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2521 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2581 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2641 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2701 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2761 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2821 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     2881 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2941 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3001 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3061 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3121 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3181 tgacagttag aaaaactcat cgagcat
//
