LOCUS       sequence-68789-e        3654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to express a CRISPR guide RNA in C. elegans from U6 
            regulatory sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3654)
  AUTHORS   Waaijers S, Portegijs V, Kerver J, Lemmens BB, Tijsterman M, van den
            Heuvel S, Boxem M
  TITLE     CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans.
  JOURNAL   Genetics. 2013 Aug 26.
  PUBMED    23979586
REFERENCE   2  (bases 1 to 3654)
  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: "Genetics. 
            2013 Aug 26."
COMMENT     Sequence Label: pMB70
FEATURES             Location/Qualifiers
     source          1..3654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        214..289
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1227..1246)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1228..1246)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1253..1270)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1253..1269)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1262..1284)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1393..1410)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1503..2091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1583..1602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2265..3059)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(3060..3164)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    3461..3482
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3497..3502,3503..3520,3521..3527)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3535..3551
                     /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     3540..3562
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3559..3575
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3596..3614
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     join(3651..3654,1..13)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3651..3654,1..13)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 gaactagtgg atcccccggg ctgcaggaat tcgatgagct cccaacacat agtgtttcca
       61 atgttatacc caatcaataa tagcaagtca ataaactacc tctacactat ttctggtaat
      121 aggcgaaacc tctacactgt cagtcacttt gtaaggtgtg cctatatatt tccataatat
      181 ttcatacaaa ttagagaccg agagagggtc tcagttttag agctagaaat agcaagttaa
      241 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttatca
      301 agcttctgac atagagtttt acatatatct tctctgtatt gtttcaattt tagtatatgt
      361 tctcgaaaga aatactctta aaataatcta attttaccaa gagccataat tcaacacttc
      421 aaaaagttag tagttgttct aaattgtcag ctacattttt ggcgaattgc caaatttctg
      481 aaaaaatagt ttatagattt gttgccaact aatttctgaa ttttgatcta ccgtaatgct
      541 tataacatat ggagcgccag tgggaaaact gttggaaacg ctgtaatagt gctaacatga
      601 ccaaacaaca tagtgaaaga gacaccacga attcaagatc tggccgaatt acggtttccg
      661 gatttgacac tgttccagat ccggaaatct accagatctt gaatccgtgg tgtctctagt
      721 gaaacttttc ataaaaactt ttattgtagt gttgtaaaca tatgagagtg cttaagctac
      781 tcaaaaatac ccattttccc gtaaatttgg aaatatacca gaaaaaattt tcaaaagcca
      841 aatcattttc gagtcacgat aattggtaat tttggattaa taatgtattt agacaacttt
      901 cttactgccg ccactccatc ttaaaataca aagtttatac gaaaactgca tgtttcaaaa
      961 ctgattggaa tctttttgaa atctgtgcat aaaagtcagt acaaaaccaa tcaaaagtag
     1021 aagatatttc aaagctcatc catcagaaga gctcatccag aagacgtttc tcttttccta
     1081 tctacgtcat catctaacac gttctcaaca tctttacgct ctccttgtct cgtatactct
     1141 ataggttttc atggcttatt ctggcatggt gccaattttt ctgtgctctt cggtcgacct
     1201 cgaggggggg cccggtaccc aattcgccct atagtgagtc gtattacaat tcactggccg
     1261 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     1321 cacatccccc tttcgccagc tgcattaaca tggtcatagc tgtttccttg cgtattgggc
     1381 gctctccgct tcctcgctca ctgactcgct gcgctcggtc gttcgggtaa agcctggggt
     1441 gcctaatgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1501 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1561 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1621 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1681 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1741 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1801 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1861 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1921 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     1981 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2041 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2101 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2161 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2221 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttattagaa aaattcatcc
     2281 agcagacgat aaaacgcaat acgctggcta tccggtgccg caatgccata cagcaccaga
     2341 aaacgatccg cccattcgcc gcccagttct tccgcaatat cacgggtggc cagcgcaata
     2401 tcctgataac gatccgccac gcccagacgg ccgcaatcaa taaagccgct aaaacggcca
     2461 ttttccacca taatgttcgg caggcacgca tcaccatggg tcaccaccag atcttcgcca
     2521 tccggcatgc tcgctttcag acgcgcaaac agctctgccg gtgccaggcc ctgatgttct
     2581 tcatccagat catcctgatc caccaggccc gcttccatac gggtacgcgc acgttcaata
     2641 cgatgtttcg cctgatgatc aaacggacag gtcgccgggt ccagggtatg cagacgacgc
     2701 atggcatccg ccataatgct cactttttct gccggcgcca gatggctaga cagcagatcc
     2761 tgacccggca cttcgcccag cagcagccaa tcacggcccg cttcggtcac cacatccagc
     2821 accgccgcac acggaacacc ggtggtggcc agccagctca gacgcgccgc ttcatcctgc
     2881 agctcgttca gcgcaccgct cagatcggtt ttcacaaaca gcaccggacg accctgcgcg
     2941 ctcagacgaa acaccgccgc atcagagcag ccaatggtct gctgcgccca atcatagcca
     3001 aacagacgtt ccacccacgc tgccgggcta cccgcatgca ggccatcctg ttcaatcata
     3061 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3121 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3181 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3241 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3301 agaccgagat agggttgagt ggccgctaca gggcgctccc attcgccatt caggctgcgc
     3361 aactgttggg aagggcgttt cggtgcgggc ctcttcgcta ttacgccagc tggcacgaca
     3421 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     3481 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     3541 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcgaaatta
     3601 accctcacta aagggaacaa aagctggagc tccaccgcgg tggcggccgc tcta
//
