LOCUS       sequence-423154-        3401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for Cloning of multiple gRNAs driven by distinct promoters 
            (H1 and mU6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3401)
  TITLE     Multiple gRNA Cloning Vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3401)
  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            "Unpublished"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-H1-Esp3I-mU6-Kan
FEATURES             Location/Qualifiers
     source          1..3401
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..39)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      933..1521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1422..1441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1675..1692
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1809..1830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1845..1850,1851..1868,1869..1875)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1883..1899
                     /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)."
     primer_bind     1888..1910
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1907..1923
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1907..1923
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        complement(1980..2293)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(2041..2061)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        complement(2300..2375)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2404..2618)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(2464..2483)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(2753..2770)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2753..2769)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2762..2784)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2978..2997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3097..3119
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3157..3175)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3243..3347
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3348..3401,1..762)
                     /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 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
       61 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
      121 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
      181 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
      241 actgcgatcc ctgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
      301 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
      361 tgtcctttta acagcgatcg cgtatttcgt ttcgctcagg cgcaatcacg aatgaataac
      421 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
      481 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
      541 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
      601 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
      661 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
      721 aataaattgc agtttcattt gatgctcgat gagtttttct aactgtcaga ccaagtttac
      781 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
      841 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
      901 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
      961 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     1021 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     1081 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     1141 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     1201 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     1261 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     1321 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     1381 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     1441 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     1501 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     1561 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     1621 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     1681 tcagtgagcg aggaagcgga acagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     1741 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     1801 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     1861 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     1921 gaccatgctc gagccaagct cggcgcgcca ttgggatggt accgaattct cgaagaccca
     1981 acaaggcttt tctcgcatgc atatttatag tctcaaaaca cacaattact ttacagttag
     2041 ggtgagtttc cttttgtgct gttttttaaa ataataattt agtatttgta tctcttatag
     2101 aaatccaagc ctatcatgta aaatgtagct agtattaaaa agaacagatt atctgtcttt
     2161 tatcgcacat taagcctcta tagttactag gaaatattat atgcaaatta accggggcag
     2221 gggagtagcc gagcttctcc cacaagtctg tgcgaggggg ccggcgcggg cctagagatg
     2281 gcggcgtcgg atcaaaaaag caccgactcg gtgccacttt ttcaagttga taacggacta
     2341 gccttatttt aacttgctat ttctagctct aaaactgaga cgatatatcg tctccggtga
     2401 agagtggtct catacagaac ttataagact cccaaatcca aagacatttc acgtttatgg
     2461 tgatttccca gaacacatag cgacatgcaa atattgcagg gcgccactcc cctgtccctc
     2521 acagccatct tcctgccagg gcgcacgcgc gctgggtgtt cccgcctagt gacactgggc
     2581 ccgcgattcc ttggagcggg ttgatgacgt cagcgttcaa aaaagcaccg actcggtgcc
     2641 actttttcaa gttgataacg gactagcctt atttaaactt gctatgctgt ttccagcata
     2701 gctcttaaac aggtcttctc tctagaaccg gtatcccaat acgcgtcaat tcactggccg
     2761 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2821 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2881 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     2941 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     3001 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3061 tcccggcatc cgcttacaga caagctgtga cagtctccgg gagctgcatg tgtcagaggt
     3121 tttcaccgtc atcaccgaaa cgcgcgagac aaaagggcct cgtgatacgc ctatttttat
     3181 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3241 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3301 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agccatattc
     3361 aacgggaaac gtcttgctct aggccgcgat taaattccaa c
//
