LOCUS       sequence-99433-e        4001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for cloning U6 promoter to generate new PU6::sgRNAs by 
            PCR-fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4001)
  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 4001)
  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: pJW1310
FEATURES             Location/Qualifiers
     source          1..4001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(67..87)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(95..111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        132..481
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     primer_bind     complement(552..571)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(553..571)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(578..594)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             732..1034
                     /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     913..932
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1383..2177
                     /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             1383..2177
                     /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(1437..1456)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2047..2066
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2378..2387
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2384..2758
                     /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      2896..3484
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3385..3404
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3638..3655
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3772..3793
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3808..3813,3814..3831,3832..3838)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3846..3862
                     /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     3851..3873
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3870..3886
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3870..3886
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3904..3922
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3904..3921
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 attgtgttcg ttgagtgacc caacaaaaag aggaaacttt attgtgccgc caagaaaaaa
       61 gtctcacaac tttattatac atagttgata attcactggc cgtcgtttta caacgtcgtg
      121 actgggaaaa ccaaaaaaaa ctagcaataa aggaataaaa aactgtacac cttaaaggcg
      181 cacactctgt tttgcaaatt ttatttttag ttgtgaattt tctgctgaga cctgaaaata
      241 gcaactttag tactactata atttgtcaac cttttcaaaa aaagcatgca atttttgaga
      301 aactcttata aaagctatta ttaaaaaaac accttttttc caaaattatt ccacaaaaaa
      361 tatgttatga aatgcctaca ccctctcaca cacactcttt atactactct gtcaaactca
      421 cgagatgtct gccgcctctt gtgttgcccc tatataaaca cctcctattg cgagatgtct
      481 tgaagggcga attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag
      541 ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc
      601 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
      661 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
      721 tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg
      781 tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc cccctggcca
      841 gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg
      901 atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg
      961 aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt
     1021 tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc tagatccttt
     1081 tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg
     1141 ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta
     1201 catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg
     1261 gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttctcgccgc
     1321 caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga ggatcgtttc
     1381 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     1441 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     1501 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     1561 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     1621 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     1681 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     1741 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     1801 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     1861 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     1921 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     1981 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     2041 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     2101 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     2161 ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat tttctcctta
     2221 cgcatctgtg cggtatttca caccgcatac aggtggcact tttcggggaa atgtgcgcgg
     2281 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2341 accctgataa atgcttcaat aatagcacgt gaggagggcc accatggcca agttgaccag
     2401 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
     2461 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
     2521 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
     2581 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
     2641 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
     2701 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
     2761 cgtgctaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2821 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2881 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2941 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3001 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3061 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3121 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3181 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3241 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3301 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3361 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3421 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3481 gaaaaacgcc agcaacgcgg cctttttacg gttcctgggc ttttgctggc cttttgctca
     3541 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3601 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3661 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     3721 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     3781 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     3841 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     3901 gctatttagg tgacactata gaatactcaa gctatgcatc aagcttggta ccgagctcgg
     3961 atccactagt aacggccgcc agtgtgctgg aattcgccct t
//
