LOCUS       sequence-370780-        3707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ric-4 sgRNA expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3707)
  AUTHORS   Schwartz ML, Davis MW, Rich MS, Jorgensen EM
  TITLE     High-efficiency CRISPR gene editing in C. elegans using Cas9 
            integrated into the genome.
  JOURNAL   PLoS Genet. 2021 Nov 8;17(11):e1009755. doi: 
            10.1371/journal.pgen.1009755. eCollection 2021 Nov.
  PUBMED    34748534
REFERENCE   2  (bases 1 to 3707)
  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: "PLoS 
            Genet."; date: "2021-11-8"; volume: "17(11):e1009755. doi"; pages: "
            10.1371/journal.pgen.1009755. eCollection 2021 Nov"
COMMENT     Sequence Label: pMLS1272
FEATURES             Location/Qualifiers
     source          1..3707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     677..693
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     677..693
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        965..1314
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     misc_RNA        1336..1411
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1471..1487)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1472..1488)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1517..1537)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1517..1535)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1556..1572)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1556..1572)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1569..1591)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1580..1596
                     /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(1604..1610,1611..1628,1629..1634))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1649..1670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1789..1806)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1960..2548)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2040..2059)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2719..3510,3511..3579))
                     /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     3342..3361
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3580..3684)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgatgtggt
      721 gacatgtgta actgcatttt ttagagacaa gttagggtga aaaaacgtta cttttgtatt
      781 gggtgtttaa aaattgaaaa gcgcttttaa tccatagcca gatttggttt ttttaataag
      841 tgctgaaaat gtgtgtgcct ttaaaaagaa cagtagtcgt aattatgttt tcttgctttt
      901 ctaaattttg caggttaatc aatttttttc acatgaaata aaatatattt tcccaaaaat
      961 ttaacaaaaa aaactagcaa taaaggaata aaaaactgta caccttaaag gcgcacactc
     1021 tgttttgcaa attttatttt tagttgtgaa ttttctgctg agacctgaaa atagcaactt
     1081 tagtactact ataatttgtc aaccttttca aaaaaagcat gcaatttttg agaaactctt
     1141 ataaaagcta ttattaaaaa aacacctttt ttccaaaatt attccacaaa aaatatgtta
     1201 tgaaatgcct acaccctctc acacacactc tttatactac tctgtcaaac tcacgagatg
     1261 tctgccgcct cttgtgtccc ccctatataa acacctccta ttgcgagatg tcttgagagg
     1321 ctggaatcaa aacttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     1381 tcaacttgaa aaagtggcac cgagtcggtg cttttttttg tgaaattttc tataattttt
     1441 tttgcagaca tgtgtggtga tcaagcttat cgataccgtc gacctcgagg gggggcccgg
     1501 tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     1561 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggga
     1621 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1681 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1741 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcata tgcgcttcct cgctcactga
     1801 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     1861 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     1921 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     1981 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2041 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2101 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2161 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2221 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2281 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2341 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2401 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2461 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2521 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2581 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2641 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2701 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2761 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     2821 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     2881 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     2941 tttatctgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3001 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3061 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3121 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3181 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3241 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3301 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3361 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3421 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3481 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3541 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     3601 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     3661 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
