LOCUS       sequence-146473-        4848 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid that serves as the PCR template to create the genomic pdt1 
            insertions contained in the EPD library. Plasmid contains the R6K 
            origin of replication so needs pir gene to replicate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4848)
  AUTHORS   Cameron DE, Collins JJ
  TITLE     Tunable protein degradation in bacteria.
  JOURNAL   Nat Biotechnol. 2014 Dec;32(12):1276-81. doi: 10.1038/nbt.3053. Epub
            2014 Nov 17.
  PUBMED    25402616
REFERENCE   2  (bases 1 to 4848)
  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"; doi: "10.1038/nbt.3053"; 
            journalName: "Nat Biotechnol"; date: "2014-12"; volume: "32"; issue:
            "12"; pages: "1276-81"
COMMENT     Sequence Label: pECM1
FEATURES             Location/Qualifiers
     source          1..4848
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    282..315
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             683..1477
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(737..756)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1347..1366
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1490..1537
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     complement(2261..2280)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2419..2441)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2582..2601
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(2658..3449,3450..3518))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3281..3300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3519..3623)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3691..3709
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3733..4121
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     rep_origin      complement(4184..4639)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4321..4342)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4533..4552
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4766..4788
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4780..4797
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4781..4797
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4807..4826
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4807..4825
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctcgagaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggctcccaat
       61 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaactggc acgacaggtt
      121 tcccgactgg aaagcgggca ggttggagcg gcgaacaaaa acgaagaaaa caccaacgaa
      181 gtgccgacct ttatgctgaa cgcgggccag gcgaaccgtc ttcagttgta accgcggagg
      241 agaaagtacc atgattccgg ggatccgtcg acctgcagtt cgaagttcct attctctaga
      301 aagtatagga acttcagagc gcttttgaag ctcacgctgc cgcaagcact cagggcgcaa
      361 gggctgctaa aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg
      421 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca
      481 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag
      541 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa
      601 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga
      661 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
      721 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
      781 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
      841 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
      901 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
      961 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     1021 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     1081 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     1141 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     1201 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     1261 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     1321 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     1381 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     1441 catcgccttc tatcgccttc ttgacgagtt cttctaataa ggggatcttg aagttcctat
     1501 tccgaagttc ctattctcta gaaagtatag gaacttcgaa gcagctccag cctacatccg
     1561 actaaacgcg taaaacagcc agcgctggcg cgatttagcc ccgacatagc cccactgttc
     1621 gtccatttcc gcgcagacga tgacgtcact gcccggctgt atgcgcgagg ttaccgactg
     1681 cggcctgagt tttttaagtg acgtaaaatc gtgttgaggc caacgcccat aatgcgggct
     1741 gttgcccggc atccaacgcc attcatggcc atatcaatga ttttctggtg cgtaccgggt
     1801 tgagaagcgg tgtaagtgaa ctgcataaac cagccattga gtaagttttt aagcacatca
     1861 tcatcataag ctttaagttg gttctcttgg atcaatttgc tgacaatggc gtttacctta
     1921 ccagtaatgt attcaaggct aattttttca agttcattcc aaccaatgat aggcatcact
     1981 tcttggatag ggataaggtt tttattatta tcaataatat aatcaagata atgttcaaat
     2041 atactttcta aggcagacca accatttgtt aaatcagttt ttgttgtgat gtaggcatca
     2101 atcataatta attgctgctt ataacaggca ctgagtaatt gttttttatt tttaaagtga
     2161 tgataaaagg cacctttggt caccaacgct tttcccgaga tcctctacgc cggacgcatc
     2221 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     2281 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     2341 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgctgcc tcgcgcgttt
     2401 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     2461 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     2521 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     2581 gcggcatcag agcagattgt actgagagtg caccataaat caatctaaag tatatatgag
     2641 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2701 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2761 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     2821 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2881 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2941 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     3001 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3061 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3121 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3181 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3241 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
     3301 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3361 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3421 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3481 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3541 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3601 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     3661 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt
     3721 caagaattcc catgtcagcc gttaagtgtt cctgtgtcac tcaaaattgc tttgagaggc
     3781 tctaagggct tctcagtgcg ttacatccct ggcttgttgt ccacaaccgt taaaccttaa
     3841 aagctttaaa agccttatat attctttttt ttcttataaa acttaaaacc ttagaggcta
     3901 tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag tacgtgaaac
     3961 atgagagctt agtacgttag ccatgagagc ttagtacgtt agccatgagg gtttagttcg
     4021 ttaaacatga gagcttagta cgttaaacat gagagcttag tacgtgaaac atgagagctt
     4081 agtacgtact atcaacaggt tgaactgctg atcttcagat cctctacgcc ggacgcatcg
     4141 tggggatctg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     4201 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4261 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     4321 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     4381 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     4441 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     4501 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     4561 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     4621 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     4681 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     4741 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
     4801 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggcccccc
//
