LOCUS       sequence-122661-        3862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gly-Ser-4xGly linker-degron-TEV-3xFLAG with 40 bp nhr-25 homology 
            arms.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3862)
  AUTHORS   Zhang L, Ward JD, Cheng Z, Dernburg AF
  TITLE     The auxin-inducible degradation (AID) system enables versatile 
            conditional protein depletion in C. elegans.
  JOURNAL   Development. 2015 Nov 9. pii: dev.129635.
  PUBMED    26552885
REFERENCE   2  (bases 1 to 3862)
  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: 
            "Development. 2015 Nov 9. pii: dev.129635."
COMMENT     Sequence Label: pJW1354
FEATURES             Location/Qualifiers
     source          1..3862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..18
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2..18
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     25..44
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        25..43
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             197..328
                     /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             329..349
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             356..421
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        complement(536..554)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(537..554)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(572..588)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(572..588)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(585..607)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    596..612
                     /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)."
     promoter        complement(join(620..626,627..644,645..650))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    665..686
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(803..820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(974..1562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1054..1073)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1700..2074)
                     /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"
     regulatory      2071..2080
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2281..3075)
                     /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             complement(2281..3075)
                     /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(2392..2411)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3002..3021
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3424..3726)
                     /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     complement(3526..3545)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     join(3849..3862,1..9)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt gggccctcta
       61 gatgcatgct cgagcggccg ccagtgtgat ggatatctgc agaattcgcc ctttcccagc
      121 aatccaactg ccaaccccac aagccacata taccgcagta ccttatatgg catcaggatc
      181 cggaggtggc gggatgccta aagatccagc caaacctccg gccaaggcac aagttgtggg
      241 atggccaccg gtgagatcat accggaagaa cgtgatggtt tcctgccaaa aatcaagcgg
      301 tggcccggag gcggcggcgt tcgtgaagga gaatctgtac tttcaatccg gaaaggacta
      361 caaagaccat gacggtgatt ataaagatca tgatatcgat tacaaggatg acgatgacaa
      421 gtaatgtctc tgtcatagga gctgaaaact tccaataagg gcgaattcca gcacactggc
      481 ggccgttact agtggatccg agctcggtac caagcttgat gcatagcttg agtattctat
      541 agtgtcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
      601 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
      661 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
      721 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
      781 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      841 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      901 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaagcc caggaaccgt aaaaaggccg
      961 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1021 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1081 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1141 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1201 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1261 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1321 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1381 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     1441 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     1501 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1561 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     1621 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     1681 aatgaagttt tagcacgtgt cagtcctgct cctcggccac gaagtgcacg cagttgccgg
     1741 ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg gtcatggccg
     1801 gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg tacagctcgt
     1861 ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg tcctggaccg
     1921 cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc tccacgaagt
     1981 cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg tcgcgcgcgg
     2041 tgagcaccgg aacggcactg gtcaacttgg ccatggtggc cctcctcacg tgctattatt
     2101 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2161 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgta tgcggtgtga
     2221 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaataat
     2281 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     2341 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     2401 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     2461 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     2521 gacgagatcc tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc
     2581 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     2641 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     2701 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     2761 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     2821 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     2881 cgctgcctcg tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     2941 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     3001 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     3061 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagagc ttgatcccct
     3121 gcgccatcag atccttggcg gcgagaaagc catccagttt actttgcagg gcttcccaac
     3181 cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca
     3241 gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt
     3301 ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg
     3361 actggctttc tacgtgaaaa ggatctaggt gaagatcctt tttgataatc tcatgcctga
     3421 catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct
     3481 gagatcagcc acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat
     3541 catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc
     3601 tgacagcaga cgtgcactgg ccagggggat caccatccgt cgccccggcg tgtcaataat
     3661 atcactctgt acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt
     3721 aaactgccgt acgtataggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     3781 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     3841 agggttttcc cagtcacgac gt
//
