LOCUS       sequence-99434-e        4002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for cloning sgRNA(F+E) to generate new PU6::sgRNAs by 
            PCR-fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4002)
  AUTHORS   Ward JD
  TITLE     Rapid and Precise Engineering of the Caenorhabditis elegans Genome 
            with Lethal Mutation Co-conversion and Inactivation of NHEJ Repair.
  JOURNAL   Genetics. 2014 Dec 9. pii: genetics.114.172361.
  PUBMED    25491644
REFERENCE   2  (bases 1 to 4002)
  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. 
            2014 Dec 9. pii: genetics.114.172361."
COMMENT     Sequence Label: pJW1311
FEATURES             Location/Qualifiers
     source          1..4002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    5..21
                     /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     10..32
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     29..45
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     29..45
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        63..81
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     63..80
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     164..182
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(220..242)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(713..732)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(714..732)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(739..756)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(739..755)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(748..770)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             893..1195
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1074..1093
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1544..2338
                     /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"
     CDS             1544..2338
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1598..1617)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2208..2227
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2539..2548
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2545..2919
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      3057..3645
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3546..3565
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3799..3816
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3933..3954
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3969..3974,3975..3992,3993..3999)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
       61 ctatttaggt gacactatag aatactcaag ctatgcatca agcttggtac cgagctcgga
      121 tccactagta acggccgcca gtgtgctgga attcgccctt aaaaataggc gtatcacgag
      181 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
      241 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
      301 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgc
      361 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
      421 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
      481 ctcttcgcta ttacgccaga aatttcacaa aaaaaagcac cgactcggtg ccactttttc
      541 aagttgataa cggactagcc ttatttaaac ttgctatgct gtttccagca tagctcttaa
      601 actcagggtt tcagcctcgt tcaagacatc tcgcaatagg aggaagggcg aattctgcag
      661 atatccatca cactggcggc cgctcgagca tgcatctaga gggcccaatt cgccctatag
      721 tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
      781 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
      841 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctatacgtac ggcagtttaa
      901 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
      961 tattgacacg ccggggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
     1021 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat
     1081 gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag
     1141 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc
     1201 aggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga aagccagtcc
     1261 gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga caagggaaaa
     1321 cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg
     1381 ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt
     1441 tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct gatggcgcag
     1501 gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     1561 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     1621 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     1681 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     1741 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     1801 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     1861 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     1921 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     1981 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     2041 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     2101 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     2161 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     2221 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     2281 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat
     2341 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2401 acaccgcata caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2461 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2521 taatagcacg tgaggagggc caccatggcc aagttgacca gtgccgttcc ggtgctcacc
     2581 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
     2641 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
     2701 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
     2761 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
     2821 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
     2881 ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctaaa acttcatttt
     2941 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3001 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3061 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3121 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3181 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3241 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3301 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3361 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3421 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3481 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3541 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3601 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3661 gcctttttac ggttcctggg cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3721 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3781 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     3841 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3901 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     3961 ccccaggctt tacactttat gcttccggct cgtatgttgt gt
//
