LOCUS       sequence-320590-        3615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR repair template: add insertion site specific homology arms by
            PCR before insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3615)
  AUTHORS   Ashley GE, Duong T, Levenson MT, Martinez MAQ, Johnson LC, Hibshman 
            JD, Saeger HN, Palmisano NJ, Doonan R, Martinez-Mendez R, Davidson 
            BR, Zhang W, Ragle JM, Medwig-Kinney TN, Sirota SS, Goldstein B, 
            Matus DQ, Dickinson DJ, Reiner DJ, Ward JD
  TITLE     An expanded auxin-inducible degron toolkit for Caenorhabditis 
            elegans.
  JOURNAL   Genetics. 2021 Mar 31;217(3). pii: 6104563. doi: 
            10.1093/genetics/iyab006.
  PUBMED    33677541
REFERENCE   2  (bases 1 to 3615)
  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.";
            date: "2021-03-31"; volume: "217(3). pii: 6104563. doi"; pages: " 
            10.1093/genetics/iyab006"
COMMENT     Sequence Label: pJW2171
FEATURES             Location/Qualifiers
     source          1..3615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..106
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(265..292)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(384..470)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     519..541
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     533..550
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     534..550
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1533..1553
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1575..1706
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     CDS             1734..1799
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1853..1872)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1854..1872)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1877..1893)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1877..1893)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1995..2014)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2006..2815
                     /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(2073..2092)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2962..3550
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3451..3470
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
       61 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
      121 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
      181 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc gtaccgctag
      241 ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt
      301 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca
      361 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
      421 cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca
      481 gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac gttgtaaaac
      541 gacggccagt cttaagctcg ggccccggtg gcggtggatc gggaggagga ggttcgggtg
      601 gcggaggcag tggaggtggc ggaagtggcg gcggtggttc aggaggaggc ggatccatgg
      661 tctccaaggg agaggaggac aacatggcct ccctcccagc cacccacgag ctccacatct
      721 tcggatccat caacggagtc gacttcgaca tggtcggaca aggaaccgga aacccaaacg
      781 acggatacga ggagctcaac ctcaagtcca ccaaggtaag tttaaacata tatatactaa
      841 ctaaccctga ttatttaaat tttcagggag acctccaatt ctccccatgg atcctcgtcc
      901 cacacatcgg atacggattc caccaatacc tcccataccc agacggaatg tccccattcc
      961 aagccgccat ggtcgacgga tccggatacc aagtccaccg taccatgcaa ttcgaggacg
     1021 gagcctccct caccgtcaac taccgttaca cctacgaggg atcccacatc aaggtaagtt
     1081 taaacagttc ggtactaact aaccatacat atttaaattt tcagggagag gcccaagtca
     1141 agggaaccgg attcccagcc gacggaccag tcatgaccaa ctccctcacc gccgccgact
     1201 ggtgccgttc caagaagacc tacccaaacg acaaggtaag tttaaacatg attttactaa
     1261 ctaactaatc tgatttaaat tttcagacca tcatctccac cttcaagtgg tcctacacca
     1321 ccggaaacgg aaagcgttac cgttccaccg cccgtaccac ctacaccttc gccaagccaa
     1381 tggccgccaa ctacctcaag aaccaaccaa tgtacgtctt ccgtaagacc gagctcaagc
     1441 actccaagac cgagctcaac ttcaaggagt ggcaaaaggc cttcaccgac gtcatgggaa
     1501 tggacgagct ctacaagggc ggcagcggag gagagaatct gtactttcaa tccggatccg
     1561 gaggtggcgg gatgcctaaa gatccagcca aacctccggc caaggcacaa gttgtgggat
     1621 ggccaccggt gagatcatac cggaagaacg tgatggtttc ctgccaaaaa tcaagcggtg
     1681 gcccggaggc ggcggcgttc gtgaagggtg gtggcggttc tggctcagga tctgactaca
     1741 aagaccatga cggtgattat aaagatcatg atatcgatta caaggatgac gatgacaagg
     1801 gagcatcggg agcctcagga gcatcgtccg gaggcgccat ccagctgata tcccctatag
     1861 tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct
     1921 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     1981 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     2041 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     2101 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     2161 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     2221 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     2281 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     2341 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     2401 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     2461 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     2521 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     2581 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     2641 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     2701 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     2761 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     2821 attggttaat tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag
     2881 ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt
     2941 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3001 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3061 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3121 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3181 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3241 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3301 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3361 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3421 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3481 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3541 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3601 tgctcacatg ttctt
//
