LOCUS       sequence-125217-        4463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA(F+E) targeting pha-1 (no Cas9); for co-conversion in C. 
            elegans.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4463)
  AUTHORS   Ward J
  TITLE     Ward lab CRISPR plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4463)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pJW1328
FEATURES             Location/Qualifiers
     source          1..4463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..50
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     32..49
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    complement(196..216)
                     /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(224..240)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        261..610
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans R07E5.16 U6 snRNA gene 
                     (Dickinson et al., 2013)"
     primer_bind     992..1014
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1052..1070)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1143..1162)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1144..1162)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1169..1185)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1323..1625
                     /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     1504..1523
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1974..2768
                     /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             1974..2768
                     /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(2028..2047)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2638..2657
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2969..2978
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2975..3349
                     /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      3487..4075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3976..3995
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4229..4246
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4363..4384
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4399..4404,4405..4422,4423..4429)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4437..4453
                     /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     join(4442..4463,1)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(4461..4463,1..14)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(4461..4463,1..14)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gaaacagcta tgaccatgat tacgccaagc tatttaggtg acactataga atactcaagc
       61 tatgcatcaa gcttggtacc gagctcggat ccactagtaa cggccgccag tgtgctggaa
      121 ttcgccctta ttgtgttcgt tgagtgaccc aacaaaaaga ggaaacttta ttgtgccgcc
      181 aagaaaaaag tctcacaact ttattataca tagttgataa ttcactggcc gtcgttttac
      241 aacgtcgtga ctgggaaaac caaaaaaaac tagcaataaa ggaataaaaa actgtacacc
      301 ttaaaggcgc acactctgtt ttgcaaattt tatttttagt tgtgaatttt ctgctgagac
      361 ctgaaaatag caactttagt actactataa tttgtcaacc ttttcaaaaa aagcatgcaa
      421 tttttgagaa actcttataa aagctattat taaaaaaaca ccttttttcc aaaattattc
      481 cacaaaaaat atgttatgaa atgcctacac cctctcacac acactcttta tactactctg
      541 tcaaactcac gagatgtctg ccgcctcttg tgttgcccct atataaacac ctcctattgc
      601 gagatgtctt gatgaataac ttgatgaaca tgtttaagag ctatgctgga aacagcatag
      661 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      721 tttttgtgaa atttctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      781 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
      841 ggtatttcac accgcatatg gtgcactctc agtgcaatct gctctgatgc cgcatagtta
      901 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
      961 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     1021 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttaagggcg
     1081 aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga gggcccaatt
     1141 cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg
     1201 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     1261 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctatacgtac
     1321 ggcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac
     1381 agagtgatat tattgacacg ccggggcgac ggatggtgat ccccctggcc agtgcacgtc
     1441 tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct
     1501 ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg
     1561 ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa
     1621 tataaatgtc aggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga
     1681 aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
     1741 caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat
     1801 agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct
     1861 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct
     1921 gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg
     1981 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     2041 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     2101 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     2161 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     2221 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     2281 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     2341 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     2401 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     2461 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     2521 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     2581 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     2641 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     2701 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     2761 tcttctgaat tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt
     2821 gcggtatttc acaccgcata caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2881 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2941 aatgcttcaa taatagcacg tgaggagggc caccatggcc aagttgacca gtgccgttcc
     3001 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt
     3061 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt
     3121 catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg
     3181 cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc
     3241 ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg
     3301 cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctaaa
     3361 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3421 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3481 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3541 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3601 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3661 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3721 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3781 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3841 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3901 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3961 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4021 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4081 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4141 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4201 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4261 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4321 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     4381 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     4441 gagcggataa caatttcaca cag
//
