LOCUS       sequence-146476-        4534 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid that serves as the PCR template to create genomic pdt1D 
            insertions. 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 4534)
  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 4534)
  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: pECT1D
FEATURES             Location/Qualifiers
     source          1..4534
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    276..309
                     /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             677..1471
                     /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(731..750)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1341..1360
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1484..1531
                     /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(1947..1966)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2105..2127)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2268..2287
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(2344..3135,3136..3204))
                     /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     2967..2986
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3205..3309)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3377..3395
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3419..3807
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     rep_origin      complement(3870..4325)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4007..4028)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4219..4238
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4452..4474
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4466..4483
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4467..4483
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4493..4512
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4493..4511
                     /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 ggcggcgaac aaaaacgaag aaaacaccaa cgaagtggcc
      181 cccaatatgc tgaacgcggg ccaggcgaac cgtcttcagt tgtaaccgcg gaggagaaag
      241 taccatgatt ccggggatcc gtcgacctgc agttcgaagt tcctattctc tagaaagtat
      301 aggaacttca gagcgctttt gaagctcacg ctgccgcaag cactcagggc gcaagggctg
      361 ctaaaggaag cggaacacgt agaaagccag tccgcagaaa cggtgctgac cccggatgaa
      421 tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc
      481 ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag caagcgaacc
      541 ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat
      601 ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc aagagacagg
      661 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
      721 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
      781 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
      841 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
      901 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
      961 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     1021 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     1081 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     1141 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     1201 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     1261 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     1321 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     1381 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     1441 cttctatcgc cttcttgacg agttcttcta ataaggggat cttgaagttc ctattccgaa
     1501 gttcctattc tctagaaagt ataggaactt cgaagcagct ccagcctaca taaaagcttt
     1561 aagttggttc tcttggatca atttgctgac aatggcgttt accttaccag taatgtattc
     1621 aaggctaatt ttttcaagtt cattccaacc aatgataggc atcacttctt ggatagggat
     1681 aaggttttta ttattatcaa taatataatc aagataatgt tcaaatatac tttctaaggc
     1741 agaccaacca tttgttaaat cagtttttgt tgtgatgtag gcatcaatca taattaattg
     1801 ctgcttataa caggcactga gtaattgttt tttattttta aagtgatgat aaaaggcacc
     1861 tttggtcacc aacgcttttc ccgagatcct ctacgccgga cgcatcgtgg ccggcatcac
     1921 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     1981 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
     2041 ggccggggga ctgttgggcg ccatctcctt gctgcctcgc gcgtttcggt gatgacggtg
     2101 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     2161 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     2221 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
     2281 gattgtactg agagtgcacc ataaatcaat ctaaagtata tatgagtaaa cttggtctga
     2341 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2401 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2461 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2521 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2581 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2641 caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     2701 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     2761 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2821 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2881 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2941 ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt
     3001 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3061 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3121 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3181 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3241 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3301 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     3361 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattcccatg
     3421 tcagccgtta agtgttcctg tgtcactcaa aattgctttg agaggctcta agggcttctc
     3481 agtgcgttac atccctggct tgttgtccac aaccgttaaa ccttaaaagc tttaaaagcc
     3541 ttatatattc ttttttttct tataaaactt aaaaccttag aggctattta agttgctgat
     3601 ttatattaat tttattgttc aaacatgaga gcttagtacg tgaaacatga gagcttagta
     3661 cgttagccat gagagcttag tacgttagcc atgagggttt agttcgttaa acatgagagc
     3721 ttagtacgtt aaacatgaga gcttagtacg tgaaacatga gagcttagta cgtactatca
     3781 acaggttgaa ctgctgatct tcagatcctc tacgccggac gcatcgtggg gatctggttc
     3841 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     3901 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3961 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     4021 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     4081 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     4141 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     4201 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     4261 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     4321 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     4381 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     4441 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga
     4501 ctcactatag ggcgaattgg gtaccgggcc cccc
//
