LOCUS       sequence-117336-        3482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty vector for cloning sgRNA. NotI and BamHI for cloning sgRNA. 
            PU6 driven sgRNA. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3482)
  AUTHORS   Norris AD, Kim HM, Colaiacovo MP, Calarco JA
  TITLE     Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of
            Dual-Marker Selection Cassettes.
  JOURNAL   Genetics. 2015 Oct;201(2):449-58. doi: 10.1534/genetics.115.180679. 
            Epub 2015 Jul 30.
  PUBMED    26232410
REFERENCE   2  (bases 1 to 3482)
  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"; doi: 
            "10.1534/genetics.115.180679"; journalName: "Genetics"; date: 
            "2015-10"; volume: "201"; issue: "2"; pages: "449-58"
COMMENT     Sequence Label: pHKMC1: Empty sgRNA for Cloning
FEATURES             Location/Qualifiers
     source          1..3482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     55..73
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(111..133)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     233..252
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     446..468
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     460..477
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     461..477
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        484..983
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans K09B11.12 U6 snRNA gene 
                     (Friedland et al., 2013)"
     misc_RNA        1005..1080
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1088..1324
                     /label=3' CeU6
                     /note="region downstream of the C. elegans K09B11.12 U6 
                     snRNA gene"
     primer_bind     complement(1343..1359)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1343..1359)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1356..1378)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1367..1383
                     /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(1391..1397,1398..1415,1416..1421))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1436..1457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1574..1591)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1745..2333)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1825..1844)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2504..3295,3296..3364))
                     /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     3127..3146
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3365..3469)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
       61 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
      121 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
      181 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
      241 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
      301 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
      361 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
      421 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
      481 ttcctccaag aactcgtaca aaaatgctct gaagtaggtc tcgagatcaa tactgggaag
      541 acgaaagtct tgcgaaaccg attcgctgac cccagtaaag tctacttcgg tagcccttcc
      601 cccaccaccc agctcgacga cgtcgacgag tacatctacc tcggtcgtca aatcaacgcc
      661 caaaacaact tgatgccgga aatccaccga agacgtcgag cagcctgggc tgcattcaat
      721 ggaatcaaga atgccaccga ctccatcacc gacaagaaga ttcgtgcgaa tctgttcgac
      781 tcaattgtcc ttccagcgct cacctacgtt tcagaagcct ggacattcac caaagctcta
      841 tccgaacgag tacgaatcac acatgcctcc cccacaaatt attgatctac atccccattt
      901 ggtatagtgt cttacaggac tctcaatatg tcagctgcat gataaatggt ccctatataa
      961 ttgaattgca aatctaaatg tttgcggccg cttaccggat ccatgtttta gagctagaaa
     1021 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     1081 tttttttcaa taatattcta gatatccctt tttgaaacta aaatgaaata ctctaaattt
     1141 ttgagttcaa tgtttcaact aatcttggtt aaaggtaaat tcccggaggg acgattttaa
     1201 agttgccaga gttgattaaa ttggtccaaa aaattttatt aggaaactcg aaaaaaaatt
     1261 gatttttttt gacaatattc caaagaaaat tacaaaatct cgacgccaaa catagtcggc
     1321 tgtgaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     1381 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     1441 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     1501 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     1561 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     1621 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1681 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1741 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1801 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1861 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1921 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1981 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2041 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2101 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2161 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2221 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2281 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2341 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2401 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2461 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2521 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2581 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     2641 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2701 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2761 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2821 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     2881 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     2941 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3001 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3061 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3121 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3181 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3241 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3301 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3361 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3421 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     3481 ga
//
