LOCUS       sequence-65053-e        3481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  klp-12 targeting sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3481)
  AUTHORS   Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco 
            JA
  TITLE     Heritable genome editing in C. elegans via a CRISPR-Cas9 system.
  JOURNAL   Nat Methods. 2013 Jun 30. doi: 10.1038/nmeth.2532.
  PUBMED    23817069
REFERENCE   2  (bases 1 to 3481)
  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: "Nat 
            Methods. 2013 Jun 30. doi: 10.1038/nmeth.2532."
COMMENT     Sequence Label: PU6::klp-12_sgRNA
FEATURES             Location/Qualifiers
     source          1..3481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        402..901
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans K09B11.12 U6 snRNA gene 
                     (Friedland et al., 2013)"
     misc_RNA        922..997
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1005..1241
                     /label=3' CeU6
                     /note="region downstream of the C. elegans K09B11.12 U6 
                     snRNA gene"
     primer_bind     complement(1260..1276)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1260..1276)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1273..1295)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1284..1300
                     /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(1308..1314,1315..1332,1333..1338))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1353..1374
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1491..1508)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1662..2250)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1742..1761)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2421..3212,3213..3281))
                     /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     3044..3063
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3282..3386)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3454..3472
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cctccaagaa ctcgtacaaa
      421 aatgctctga agtaggtctc gagatcaata ctgggaagac gaaagtcttg cgaaaccgat
      481 tcgctgaccc cagtaaagtc tacttcggta gcccttcccc caccacccag ctcgacgacg
      541 tcgacgagta catctacctc ggtcgtcaaa tcaacgccca aaacaacttg atgccggaaa
      601 tccaccgaag acgtcgagca gcctgggctg cattcaatgg aatcaagaat gccaccgact
      661 ccatcaccga caagaagatt cgtgcgaatc tgttcgactc aattgtcctt ccagcgctca
      721 cctacgtttc agaagcctgg acattcacca aagctctatc cgaacgagta cgaatcacac
      781 atgcctcccc cacaaattat tgatctacat ccccatttgg tatagtgtct tacaggactc
      841 tcaatatgtc agctgcatga taaatggtcc ctatataatt gaattgcaaa tctaaatgtt
      901 tgatccacaa gttacaattg ggttttagag ctagaaatag caagttaaaa taaggctagt
      961 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttcaataa tattctagat
     1021 atcccttttt gaaactaaaa tgaaatactc taaatttttg agttcaatgt ttcaactaat
     1081 cttggttaaa ggtaaattcc cggagggacg attttaaagt tgccagagtt gattaaattg
     1141 gtccaaaaaa ttttattagg aaactcgaaa aaaaattgat tttttttgac aatattccaa
     1201 agaaaattac aaaatctcga cgccaaacat agtcggctgt gaagcttggc gtaatcatgg
     1261 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     1321 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     1381 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1441 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     1501 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1561 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1621 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1681 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1741 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1801 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1861 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1921 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1981 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2041 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2101 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2161 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2221 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2281 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     2341 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2401 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2461 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2521 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     2581 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2641 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2701 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2761 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2821 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2881 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2941 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3001 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3061 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3121 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3181 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3241 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3301 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3361 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     3421 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     3481 c
//
