LOCUS       sequence-136556-        6538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SapTrap 3-site Destination vector for C-terminal GFP tagging with 
            embedded Cbr-unc-119 selectable marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6538)
  AUTHORS   Schwartz ML, Jorgensen EM
  TITLE     SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification
            in Caenorhabditis elegans.
  JOURNAL   Genetics. 2016 Feb 2. pii: genetics.115.184275.
  PUBMED    26837755
REFERENCE   2  (bases 1 to 6538)
  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. 
            2016 Feb 2. pii: genetics.115.184275."
COMMENT     Sequence Label: pMLS235
FEATURES             Location/Qualifiers
     source          1..6538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        142..640
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans K09B11.12 U6 snRNA gene 
                     (Friedland et al., 2013)"
     misc_RNA        668..743
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    751..987
                     /label=3' CeU6
                     /note="region downstream of the C. elegans K09B11.12 U6 
                     snRNA gene"
     terminator      complement(1133..1160)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1252..1338)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1387..1409
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1401..1418
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1402..1418
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1434..1533
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1628..1656)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    2215..2248
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4212..4223
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    4372..4405
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     4595..4615
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(4689..4788)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4805..4824)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4806..4824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4829..4845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4829..4845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4947..4966)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4958..5767
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5025..5044)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5914..6502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6403..6422
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
       61 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
      121 gagtcagtga gcgaggaagc gtccaagaac tcgtacaaaa atgctctgaa gtaggtctcg
      181 agatcaatac tgggaagacg aaagtcttgc gaaaccgatt cgctgacccc agtaaagtct
      241 acttcggtag cccttccccc accacccagc tcgacgacgt cgacgagtac atctacctcg
      301 gtcgtcaaat caacgcccaa aacaacttga tgccggaaat ccaccgaaga cgtcgagcag
      361 cctgggctgc attcaatgga atcaagaatg ccaccgactc catcaccgac aagaagattc
      421 gtgcgaatct gttcgactca attgtccttc cagcgctcac ctacgtttca gaagcctgga
      481 cattcaccaa agctctatcc gaacgagtac gaatcacaca tgcctccccc acaaattatt
      541 gatctacatc cccatttggt atagtgtctt acaggactct caatatgtca gctgcatgat
      601 aaatggtccc tatataattg aattgcaaat ctaaatgttt gcgaagagcg gatccatggg
      661 ctcttcggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
      721 aaaaagtggc accgagtcgg tgcttttttt caataatatt ctagatatcc ctttttgaaa
      781 ctaaaatgaa atactctaaa tttttgagtt caatgtttca actaatcttg gttaaaggta
      841 aattcccgga gggacgattt taaagttgcc agagttgatt aaattggtcc aaaaaatttt
      901 attaggaaac tcgaaaaaaa attgattttt tttgacaata ttccaaagaa aattacaaaa
      961 tctcgacgcc aaacatagtc ggctgtggcc caatacgcaa accgcctctc cccgcgcgtt
     1021 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     1081 gcaacgcaat taatacgcgt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc
     1141 atccgtcagg atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc
     1201 ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg gatttgtcct
     1261 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt cttccgactg
     1321 agcctttcgt tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga
     1381 tgttttccca gtcacgacgt tgtaaaacga cggccagtct taagctcggg ccccaaataa
     1441 tgattttatt ttgactgata gtgacctgtt cgttgcaaca aattgatgag caatgctttt
     1501 ttataatgcc aactttgtac aaaaaagcag gctcgccgcg aagagcggat ccatgggctc
     1561 ttcgggcagc ggtggcagtg gaggtaccgg cggaagcggt agcaaaggag aagaactttt
     1621 cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc
     1681 tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctta aatttatttg
     1741 cactactgga aaactacctg ttccatgggt aagtttaaac atatatatac taactaaccc
     1801 tgattattta aattttcagc caacacttgt cactactttc tgttatggtg ttcaatgctt
     1861 ctcgagatac ccagatcata tgaaacggca tgactttttc aagagtgcca tgcccgaagg
     1921 ttatgtacag gaaagaacta tatttttcaa agatgacggg aactacaaga cacgtaagtt
     1981 taaacagttc ggtactaact aaccatacat atttaaattt tcaggtgctg aagtcaagtt
     2041 tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg
     2101 aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat acatcatggc
     2161 agacaaacaa aagaatggaa tcaaagttgt aagtttaaac atgattttac taacataact
     2221 tcgtataatg tatgctatac gaagttattt ctagacattc tctaatgaaa aaatctttca
     2281 gttgaaattg aaaatgagtt aaagttggag tttttattga aaacagattt ccgtgtgatt
     2341 agtgttttta gcgagtgtga caggacagcg aaaaaatata gaaacaaggg gggaactgaa
     2401 aagcttagga atgcattgaa catgagaagg ggaaggggaa ggaacaaact agacaggaat
     2461 tattggaatt taatcacatt tggagttttt tttctattcg acagaataat tatccagaac
     2521 atttttgtat taaatattta tgcatcatat gagtagtcgg ctttgttgtg catgacgagt
     2581 ttgttatcga cgaaatagaa gctgtcagaa cgagtctcgt ttggattgtt gatcatgtcg
     2641 tccactgaaa aagagattag tcttttgaat tgtacttttt agaataatga ctcactgagt
     2701 tgttgagaga gttgagggaa ctcatagata tgttcacagt tgtttcgtga attcggaata
     2761 cagaatccga attcaaagtc aaaacacttc agaaggcgat ctttgaagaa gtgacgttcg
     2821 atcattcgga aatgatggat tgggatgtct cctactttga attccacagt tgctccgacc
     2881 gttttgagtt tcaaaaagtt tggagcaaat ctataacgca cgtatcttgc cgactcctgt
     2941 ggcgactcat cattctcctg atcgttctcc ggtttggcga tctcgaaaag cacttgctca
     3001 gtgtccagat cacggatttg gaacttggtg aactcgatgt tatagatgtt cgcagatggg
     3061 gagcataaga atcctaaatt tatgttttaa actgaaatcc aaagggagca agataccttg
     3121 agtgattccc ggaagtgcta aaacgtcgtt cggagtgatt tgagctttct tcgcaagctc
     3181 cgattccgtt gtgattcctt gttcggtgct tggtggtggc cgtggcatct ggaaatatgg
     3241 aaaagttcaa caaaaagaaa agagaaaaga atgaaatcgg atatcaagag ttagttgagc
     3301 ggtttctcta gttttctgag tctcacctgc gacgggaagg tcgccgagcc gggtggaatc
     3361 gattgttgtt gctcggcttt catatcggtt tggttggaag cggctgaaaa cggaaagaag
     3421 tggaagaagg aaaagagtgt ggtgtgacag gaaaatggta attagagggt gccaaataac
     3481 cagctatatt ttgttttttt ttgaaaacat ttttaaaaag aaaaatacga taatgatatc
     3541 agatggattt ccggaaaact ggtatgaaaa atttcaacct ttttgagtac atgtaatcaa
     3601 aatacacttt gtaaattatc atttttattg aaactccacc atttttctat ttataacgct
     3661 aataatttga aaaagaaacc tgttgcgaac cgcggggtga atcccaaaaa cgaatgcgtt
     3721 ttggtggagt gattgattcg aatcgaagaa gaaaaagaag aagacgtgga atagagagct
     3781 cactcttaac cgagcagcac acaccgacag aaaaaaaaat gaaatgaatg agggtcttct
     3841 tcttcttctt cttcgaatga ttgacagaaa tgggaaaaag aggaagattg agaagggaaa
     3901 aaggaaggag aaaagaagca gaagaagacg tcagagagga gaggaacgag cggaaaagca
     3961 gcgggcgcaa gtcatagaag tagcagagct ggggagaaga agacactatc caagaaagga
     4021 atgacgagag agtatgcaaa ggggtatagg gtgcagacag aataggaaca gaataacaga
     4081 tgatgagcca agaagagttg aaaagggcga tgaatttgtc atgtaactta atttgggtca
     4141 atttgagcat gatgaattga aatcatccct tgttgggagt taataaccgg tttgttatca
     4201 gaaaccctgt aatagaaggg cgccctaact ttgagccaat tcatcccggt ttctgtcaaa
     4261 tatatcaaaa agtggtcaac tgacaaattg tttttgatat tataataaac attttatccg
     4321 ttaacaattt tcgaatactt tttacaagga cttggataaa ttggctcaaa gataacttcg
     4381 tataatgtat gctatacgaa gttattaact aatctgattt aaattttcag aacttcaaaa
     4441 ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc
     4501 caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca caatctgccc
     4561 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     4621 ctgggattac acatggcatg gatgaactat acaaatgacg aagagcggat ccatgggctc
     4681 ttcgactgac ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat
     4741 ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctga
     4801 tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt
     4861 gtctcaaaat ctctgatgtt acattgcaca agataaaata atatcatcat gaacaataaa
     4921 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     4981 gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     5041 cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc
     5101 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     5161 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     5221 tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt
     5281 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     5341 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     5401 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     5461 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     5521 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     5581 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     5641 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     5701 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     5761 tttctaatca gaattggtta attggttgta acactggcag agcattacgc tgacttgacg
     5821 ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc
     5881 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5941 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6001 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6061 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6121 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6181 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6241 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6301 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6361 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6421 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6481 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcc
//
