LOCUS       sequence-136587-        6980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SapTrap Single plasmid targeting vector for tagging Unc-32 with GFP 
            by CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6980)
  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 6980)
  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: pMLS338
FEATURES             Location/Qualifiers
     source          1..6980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..150
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    157..393
                     /label=3' CeU6
                     /note="region downstream of the C. elegans K09B11.12 U6 
                     snRNA gene"
     misc_RNA        complement(401..476)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(497..996)
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans K09B11.12 U6 snRNA gene 
                     (Friedland et al., 2013)"
     protein_bind    1099..1120
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1135..1140,1141..1158,1159..1165)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1173..1189
                     /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     1178..1200
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1197..1213
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1197..1213
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1232..1252
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1234..1252
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1424..1452)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    2011..2044
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4008..4019
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    4168..4201
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     4391..4411
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(4526..4542)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4527..4543)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(4567..4586)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4568..4586)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4596..4613)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4596..4612)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4605..4627)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4754..5209
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4841..4860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5051..5072
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5235..5339
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5340..5408,5409..6200)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5558..5577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6371..6959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6860..6879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
       61 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
      121 acgaccgagc gcagcgagtc agtgagcgag gaagcgcaca gccgactatg tttggcgtcg
      181 agattttgta attttctttg gaatattgtc aaaaaaaatc aatttttttt cgagtttcct
      241 aataaaattt tttggaccaa tttaatcaac tctggcaact ttaaaatcgt ccctccggga
      301 atttaccttt aaccaagatt agttgaaaca ttgaactcaa aaatttagag tatttcattt
      361 tagtttcaaa aagggatatc tagaatatta ttgaaaaaaa gcaccgactc ggtgccactt
      421 tttcaagttg ataacggact agccttattt taacttgcta tttctagctc taaaacaggt
      481 gatcttagag attctcaaac atttagattt gcaattcaat tatataggga ccatttatca
      541 tgcagctgac atattgagag tcctgtaaga cactatacca aatggggatg tagatcaata
      601 atttgtgggg gaggcatgtg tgattcgtac tcgttcggat agagctttgg tgaatgtcca
      661 ggcttctgaa acgtaggtga gcgctggaag gacaattgag tcgaacagat tcgcacgaat
      721 cttcttgtcg gtgatggagt cggtggcatt cttgattcca ttgaatgcag cccaggctgc
      781 tcgacgtctt cggtggattt ccggcatcaa gttgttttgg gcgttgattt gacgaccgag
      841 gtagatgtac tcgtcgacgt cgtcgagctg ggtggtgggg gaagggctac cgaagtagac
      901 tttactgggg tcagcgaatc ggtttcgcaa gactttcgtc ttcccagtat tgatctcgag
      961 acctacttca gagcattttt gtacgagttc ttggagccca atacgcaaac cgcctctccc
     1021 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     1081 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     1141 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     1201 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
     1261 gctggagctc caccgcggtg gcggccaatt cgattgggct ccattctcat tcgaaaaaat
     1321 ccttgctgaa gaacgtgaag ctgaggagaa tctcgcgggc agcggtggca gtggaggtac
     1381 cggcggaagc ggtatgagca aaggagaaga acttttcact ggagttgtcc caattcttgt
     1441 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga
     1501 tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc
     1561 atgggtaagt ttaaacatat atatactaac taaccctgat tatttaaatt ttcagccaac
     1621 acttgtcact actttctgtt atggtgttca atgcttctcg agatacccag atcatatgaa
     1681 acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt
     1741 tttcaaagat gacgggaact acaagacacg taagtttaaa cagttcggta ctaactaacc
     1801 atacatattt aaattttcag gtgctgaagt caagtttgaa ggtgataccc ttgttaatag
     1861 aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga
     1921 atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa
     1981 agttgtaagt ttaaacatga ttttactaac ataacttcgt ataatgtatg ctatacgaag
     2041 ttatttctag acattctcta atgaaaaaat ctttcagttg aaattgaaaa tgagttaaag
     2101 ttggagtttt tattgaaaac agatttccgt gtgattagtg tttttagcga gtgtgacagg
     2161 acagcgaaaa aatatagaaa caagggggga actgaaaagc ttaggaatgc attgaacatg
     2221 agaaggggaa ggggaaggaa caaactagac aggaattatt ggaatttaat cacatttgga
     2281 gttttttttc tattcgacag aataattatc cagaacattt ttgtattaaa tatttatgca
     2341 tcatatgagt agtcggcttt gttgtgcatg acgagtttgt tatcgacgaa atagaagctg
     2401 tcagaacgag tctcgtttgg attgttgatc atgtcgtcca ctgaaaaaga gattagtctt
     2461 ttgaattgta ctttttagaa taatgactca ctgagttgtt gagagagttg agggaactca
     2521 tagatatgtt cacagttgtt tcgtgaattc ggaatacaga atccgaattc aaagtcaaaa
     2581 cacttcagaa ggcgatcttt gaagaagtga cgttcgatca ttcggaaatg atggattggg
     2641 atgtctccta ctttgaattc cacagttgct ccgaccgttt tgagtttcaa aaagtttgga
     2701 gcaaatctat aacgcacgta tcttgccgac tcctgtggcg actcatcatt ctcctgatcg
     2761 ttctccggtt tggcgatctc gaaaagcact tgctcagtgt ccagatcacg gatttggaac
     2821 ttggtgaact cgatgttata gatgttcgca gatggggagc ataagaatcc taaatttatg
     2881 ttttaaactg aaatccaaag ggagcaagat accttgagtg attcccggaa gtgctaaaac
     2941 gtcgttcgga gtgatttgag ctttcttcgc aagctccgat tccgttgtga ttccttgttc
     3001 ggtgcttggt ggtggccgtg gcatctggaa atatggaaaa gttcaacaaa aagaaaagag
     3061 aaaagaatga aatcggatat caagagttag ttgagcggtt tctctagttt tctgagtctc
     3121 acctgcgacg ggaaggtcgc cgagccgggt ggaatcgatt gttgttgctc ggctttcata
     3181 tcggtttggt tggaagcggc tgaaaacgga aagaagtgga agaaggaaaa gagtgtggtg
     3241 tgacaggaaa atggtaatta gagggtgcca aataaccagc tatattttgt ttttttttga
     3301 aaacattttt aaaaagaaaa atacgataat gatatcagat ggatttccgg aaaactggta
     3361 tgaaaaattt caaccttttt gagtacatgt aatcaaaata cactttgtaa attatcattt
     3421 ttattgaaac tccaccattt ttctatttat aacgctaata atttgaaaaa gaaacctgtt
     3481 gcgaaccgcg gggtgaatcc caaaaacgaa tgcgttttgg tggagtgatt gattcgaatc
     3541 gaagaagaaa aagaagaaga cgtggaatag agagctcact cttaaccgag cagcacacac
     3601 cgacagaaaa aaaaatgaaa tgaatgaggg tcttcttctt cttcttcttc gaatgattga
     3661 cagaaatggg aaaaagagga agattgagaa gggaaaaagg aaggagaaaa gaagcagaag
     3721 aagacgtcag agaggagagg aacgagcgga aaagcagcgg gcgcaagtca tagaagtagc
     3781 agagctgggg agaagaagac actatccaag aaaggaatga cgagagagta tgcaaagggg
     3841 tatagggtgc agacagaata ggaacagaat aacagatgat gagccaagaa gagttgaaaa
     3901 gggcgatgaa tttgtcatgt aacttaattt gggtcaattt gagcatgatg aattgaaatc
     3961 atcccttgtt gggagttaat aaccggtttg ttatcagaaa ccctgtaata gaagggcgcc
     4021 ctaactttga gccaattcat cccggtttct gtcaaatata tcaaaaagtg gtcaactgac
     4081 aaattgtttt tgatattata ataaacattt tatccgttaa caattttcga atacttttta
     4141 caaggacttg gataaattgg ctcaaagata acttcgtata atgtatgcta tacgaagtta
     4201 ttaactaatc tgatttaaat tttcagaact tcaaaattag acacaacatt gaagatggaa
     4261 gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt
     4321 taccagacaa ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga
     4381 gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg
     4441 aactatacaa aggttaagat cacctcggcc acttcaaaca gtgtgacatc gacgttcgac
     4501 aaatctttaa tgtaatcaag cttatcgata ccgtcgacct cgaggggggg cccggtaccc
     4561 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     4621 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4681 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4741 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4801 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4861 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4921 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4981 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5041 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5101 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5161 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5221 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5281 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5341 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5401 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5461 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5521 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5581 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5641 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5701 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5761 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5821 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5881 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5941 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6001 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6061 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6121 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6181 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6241 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6301 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6361 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6421 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6481 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     6541 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6601 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6661 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6721 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6781 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6841 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6901 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6961 acgccagcaa cgcggccttt
//
