LOCUS       sequence-136559-        6574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SapTrap 3-site Destination vector for internal 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 6574)
  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 6574)
  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: pMLS236
FEATURES             Location/Qualifiers
     source          1..6574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     113..130
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        137..635
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans K09B11.12 U6 snRNA gene 
                     (Friedland et al., 2013)"
     misc_RNA        663..738
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    746..982
                     /label=3' CeU6
                     /note="region downstream of the C. elegans K09B11.12 U6 
                     snRNA gene"
     terminator      complement(1128..1155)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1247..1333)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1382..1404
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1396..1413
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1397..1413
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1429..1528
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1623..1651)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    2210..2243
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4207..4218
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    4367..4400
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     4590..4610
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(4720..4819)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4836..4855)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4837..4855)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4860..4876)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4860..4876)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4978..4997)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4989..5798
                     /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(5056..5075)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5945..6533
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6434..6453
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
       61 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
      121 agtgagcgag gaagcgtcca agaactcgta caaaaatgct ctgaagtagg tctcgagatc
      181 aatactggga agacgaaagt cttgcgaaac cgattcgctg accccagtaa agtctacttc
      241 ggtagccctt cccccaccac ccagctcgac gacgtcgacg agtacatcta cctcggtcgt
      301 caaatcaacg cccaaaacaa cttgatgccg gaaatccacc gaagacgtcg agcagcctgg
      361 gctgcattca atggaatcaa gaatgccacc gactccatca ccgacaagaa gattcgtgcg
      421 aatctgttcg actcaattgt ccttccagcg ctcacctacg tttcagaagc ctggacattc
      481 accaaagctc tatccgaacg agtacgaatc acacatgcct cccccacaaa ttattgatct
      541 acatccccat ttggtatagt gtcttacagg actctcaata tgtcagctgc atgataaatg
      601 gtccctatat aattgaattg caaatctaaa tgtttgcgaa gagcggatcc atgggctctt
      661 cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
      721 gtggcaccga gtcggtgctt tttttcaata atattctaga tatccctttt tgaaactaaa
      781 atgaaatact ctaaattttt gagttcaatg tttcaactaa tcttggttaa aggtaaattc
      841 ccggagggac gattttaaag ttgccagagt tgattaaatt ggtccaaaaa attttattag
      901 gaaactcgaa aaaaaattga ttttttttga caatattcca aagaaaatta caaaatctcg
      961 acgccaaaca tagtcggctg tggcccaata cgcaaaccgc ctctccccgc gcgttggccg
     1021 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     1081 gcaattaata cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg
     1141 tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
     1201 accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca
     1261 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct
     1321 ttcgttttat ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt
     1381 tcccagtcac gacgttgtaa aacgacggcc agtcttaagc tcgggcccca aataatgatt
     1441 ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata
     1501 atgccaactt tgtacaaaaa agcaggctcg ccgcgaagag cggatccatg ggctcttcgg
     1561 gcagcggtgg cagtggaggt accggcggaa gcggtagcaa aggagaagaa cttttcactg
     1621 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca
     1681 gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta
     1741 ctggaaaact acctgttcca tgggtaagtt taaacatata tatactaact aaccctgatt
     1801 atttaaattt tcagccaaca cttgtcacta ctttctgtta tggtgttcaa tgcttctcga
     1861 gatacccaga tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg
     1921 tacaggaaag aactatattt ttcaaagatg acgggaacta caagacacgt aagtttaaac
     1981 agttcggtac taactaacca tacatattta aattttcagg tgctgaagtc aagtttgaag
     2041 gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca
     2101 ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc atggcagaca
     2161 aacaaaagaa tggaatcaaa gttgtaagtt taaacatgat tttactaaca taacttcgta
     2221 taatgtatgc tatacgaagt tatttctaga cattctctaa tgaaaaaatc tttcagttga
     2281 aattgaaaat gagttaaagt tggagttttt attgaaaaca gatttccgtg tgattagtgt
     2341 ttttagcgag tgtgacagga cagcgaaaaa atatagaaac aaggggggaa ctgaaaagct
     2401 taggaatgca ttgaacatga gaaggggaag gggaaggaac aaactagaca ggaattattg
     2461 gaatttaatc acatttggag ttttttttct attcgacaga ataattatcc agaacatttt
     2521 tgtattaaat atttatgcat catatgagta gtcggctttg ttgtgcatga cgagtttgtt
     2581 atcgacgaaa tagaagctgt cagaacgagt ctcgtttgga ttgttgatca tgtcgtccac
     2641 tgaaaaagag attagtcttt tgaattgtac tttttagaat aatgactcac tgagttgttg
     2701 agagagttga gggaactcat agatatgttc acagttgttt cgtgaattcg gaatacagaa
     2761 tccgaattca aagtcaaaac acttcagaag gcgatctttg aagaagtgac gttcgatcat
     2821 tcggaaatga tggattggga tgtctcctac tttgaattcc acagttgctc cgaccgtttt
     2881 gagtttcaaa aagtttggag caaatctata acgcacgtat cttgccgact cctgtggcga
     2941 ctcatcattc tcctgatcgt tctccggttt ggcgatctcg aaaagcactt gctcagtgtc
     3001 cagatcacgg atttggaact tggtgaactc gatgttatag atgttcgcag atggggagca
     3061 taagaatcct aaatttatgt tttaaactga aatccaaagg gagcaagata ccttgagtga
     3121 ttcccggaag tgctaaaacg tcgttcggag tgatttgagc tttcttcgca agctccgatt
     3181 ccgttgtgat tccttgttcg gtgcttggtg gtggccgtgg catctggaaa tatggaaaag
     3241 ttcaacaaaa agaaaagaga aaagaatgaa atcggatatc aagagttagt tgagcggttt
     3301 ctctagtttt ctgagtctca cctgcgacgg gaaggtcgcc gagccgggtg gaatcgattg
     3361 ttgttgctcg gctttcatat cggtttggtt ggaagcggct gaaaacggaa agaagtggaa
     3421 gaaggaaaag agtgtggtgt gacaggaaaa tggtaattag agggtgccaa ataaccagct
     3481 atattttgtt tttttttgaa aacattttta aaaagaaaaa tacgataatg atatcagatg
     3541 gatttccgga aaactggtat gaaaaatttc aacctttttg agtacatgta atcaaaatac
     3601 actttgtaaa ttatcatttt tattgaaact ccaccatttt tctatttata acgctaataa
     3661 tttgaaaaag aaacctgttg cgaaccgcgg ggtgaatccc aaaaacgaat gcgttttggt
     3721 ggagtgattg attcgaatcg aagaagaaaa agaagaagac gtggaataga gagctcactc
     3781 ttaaccgagc agcacacacc gacagaaaaa aaaatgaaat gaatgagggt cttcttcttc
     3841 ttcttcttcg aatgattgac agaaatggga aaaagaggaa gattgagaag ggaaaaagga
     3901 aggagaaaag aagcagaaga agacgtcaga gaggagagga acgagcggaa aagcagcggg
     3961 cgcaagtcat agaagtagca gagctgggga gaagaagaca ctatccaaga aaggaatgac
     4021 gagagagtat gcaaaggggt atagggtgca gacagaatag gaacagaata acagatgatg
     4081 agccaagaag agttgaaaag ggcgatgaat ttgtcatgta acttaatttg ggtcaatttg
     4141 agcatgatga attgaaatca tcccttgttg ggagttaata accggtttgt tatcagaaac
     4201 cctgtaatag aagggcgccc taactttgag ccaattcatc ccggtttctg tcaaatatat
     4261 caaaaagtgg tcaactgaca aattgttttt gatattataa taaacatttt atccgttaac
     4321 aattttcgaa tactttttac aaggacttgg ataaattggc tcaaagataa cttcgtataa
     4381 tgtatgctat acgaagttat taactaatct gatttaaatt ttcagaactt caaaattaga
     4441 cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt
     4501 ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg
     4561 aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg
     4621 attacacatg gcatggatga actatacaaa ggtggcacag gaggaacggg cggtagtgga
     4681 ggcactggtc gaagagcgga tccatgggct cttcgccaga cccagctttc ttgtacaaag
     4741 ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc
     4801 aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg
     4861 tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
     4921 aagataaaat aatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     4981 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     5041 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     5101 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     5161 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     5221 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     5281 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     5341 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     5401 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     5461 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     5521 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     5581 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     5641 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     5701 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     5761 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     5821 aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc
     5881 ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5941 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6001 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6061 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6121 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6181 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6241 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6301 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6361 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6421 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6481 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6541 gcaacgcggc ctttttacgg ttcctggcct tttg
//
