LOCUS       sequence-128200-        5282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid - trimethoprim resistant donor cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5282)
  AUTHORS   Tas H, Nguyen CT, Patel R, Kim NH, Kuhlman TE
  TITLE     An Integrated System for Precise Genome Modification in Escherichia 
            coli.
  JOURNAL   PLoS One. 2015 Sep 2;10(9):e0136963. doi: 
            10.1371/journal.pone.0136963. eCollection 2015.
  PUBMED    26332675
REFERENCE   2  (bases 1 to 5282)
  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: "PLoS One.";
            date: "2015-09-2"; volume: "10(9):e0136963. doi"; pages: " 
            10.1371/journal.pone.0136963. eCollection 2015"
COMMENT     Sequence Label: pTKDP-dhfr
FEATURES             Location/Qualifiers
     source          1..5282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..77
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     62..78
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    100..133
                     /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)"
     promoter        197..696
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(232..255)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     629..648
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        708..755
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             774..1010
                     /codon_start=1
                     /gene="dhfrIIb from plasmid R388"
                     /product="E. coli plasmid-associated dihydrofolate 
                     reductase"
                     /label=TpR
                     /note="confers resistance to trimethoprim"
                     /translation="MGQSSDEANAPVAGQFALPLSATFGLGDRVRKKSGAAWQGQVVGW
                     YCTKLTPEGYAVESESHPGSVQIYPVAALERVA"
     protein_bind    1044..1077
                     /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             1358..1549
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1543..1565)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1706..1725
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             join(1872..1958,1959..3293)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     primer_bind     complement(3362..3379)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3533..4121)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3613..3632)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4292..5083,5084..5152))
                     /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     4915..4934
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5153..5257)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tagggataac agggtaatta cggccccaag gtccaaacgg tgaaggtacc gggccccccc
       61 tcgaggtcga cggtatcgat aagcttgata tcgaattccg aagttcctat tctctagaaa
      121 gtataggaac ttcaggtctg aagaggagtt tacgtccagc caagctagct tggctgcagg
      181 tcgtcgaaat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
      241 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
      301 acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct
      361 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
      421 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
      481 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
      541 cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg
      601 ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
      661 gcgctgttct cctcttcctc atctccgggc ctttcgacct gcagcctgtt gacaattaat
      721 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatgggtc
      781 aaagtagcga tgaagccaac gctcccgttg cagggcagtt tgcgcttccc ctgagtgcca
      841 cctttggctt aggggatcgc gtacgcaaga aatctggtgc cgcttggcag ggtcaagtcg
      901 tcggttggta ttgcacaaaa ctcactcctg aaggctatgc ggtcgagtcc gaatcccacc
      961 caggctcagt gcaaatttat cctgtggctg cacttgaacg tgtggcctaa tgaggggatc
     1021 aattctctag agctcgctga tcagaagttc ctattctcta gaaagtatag gaacttcgat
     1081 ggcgcctcat ccctgaagcc aatagggata acagggtaat gcaggatgct gctggctacc
     1141 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     1201 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
     1261 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     1321 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     1381 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     1441 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     1501 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     1561 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     1621 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     1681 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     1741 atgtactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact
     1801 tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg
     1861 agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta
     1921 ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat
     1981 ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac
     2041 agcaaaaaaa tgaaaaatat caagttcctg aattcgattc gtccacaatt aaaaatatct
     2101 cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg
     2161 tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg
     2221 atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga
     2281 aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa
     2341 aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt
     2401 tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag
     2461 ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa
     2521 tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact
     2581 acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca
     2641 aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt
     2701 tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac
     2761 ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg
     2821 agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag
     2881 taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca
     2941 ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc
     3001 ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg
     3061 tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac
     3121 ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg
     3181 cagacaaaca atcaacgttt gcgccaagct tcctgctgaa catcaaaggc aagaaaacat
     3241 ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taacatatgc
     3301 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     3361 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3421 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3481 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3541 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3601 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3661 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3721 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3781 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3841 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3901 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3961 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4021 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4081 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4141 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4201 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4261 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4321 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4381 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4441 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4501 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4561 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg
     4621 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4681 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4741 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4801 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4861 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     4921 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4981 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5041 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5101 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5161 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5221 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5281 ct
//
