LOCUS       sequence-128201-        6047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid - hygromycin resistant donor cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6047)
  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 6047)
  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-hph
FEATURES             Location/Qualifiers
     source          1..6047
                     /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 "
     protein_bind    1809..1842
                     /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             2123..2314
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2308..2330)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2471..2490
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             join(2637..2723,2724..4058)
                     /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(4127..4144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4298..4886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4378..4397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5057..5848,5849..5917))
                     /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     5680..5699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5918..6022)
                     /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 accatgaaaa
      781 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
      841 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
      901 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
      961 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     1021 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     1081 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     1141 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     1201 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     1261 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     1321 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     1381 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     1441 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     1501 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     1561 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     1621 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     1681 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     1741 aagtactcgc cgatagtgga aaccgacgcc ccggatgagg ggatcaattc tctagagctc
     1801 gctgatcaga agttcctatt ctctagaaag tataggaact tcgatggcgc ctcatccctg
     1861 aagccaatag ggataacagg gtaatgcagg atgctgctgg ctaccctgtg gaacacctac
     1921 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     1981 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     2041 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     2101 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     2161 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     2221 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     2281 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     2341 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2401 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     2461 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgta ctttggcgtc
     2521 accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca tcttcaaaca
     2581 ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca tgaacgatga
     2641 acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca ctgctggcag
     2701 gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag gaaacatacg
     2761 gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa aaaaatgaaa
     2821 aatatcaagt tcctgaattc gattcgtcca caattaaaaa tatctcttct gcaaaaggcc
     2881 tggacgtttg ggacagctgg ccattacaaa acgctgacgg cactgtcgca aactatcacg
     2941 gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac acatcgattt
     3001 acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac gctggccgcg
     3061 tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac caaacacaag
     3121 aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc tacactgatt
     3181 tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac gtatcagcat
     3241 cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt gacggtgacg
     3301 gaaaaacgta tcaaaatgta cagcagttca tcgatgaagg caactacagc tcaggcgaca
     3361 accatacgct gagagatcct cactacgtag aagataaagg ccacaaatac ttagtatttg
     3421 aagcaaacac tggaactgaa gatggctacc aaggcgaaga atctttattt aacaaagcat
     3481 actatggcaa aagcacatca ttcttccgtc aagaaagtca aaaacttctg caaagcgata
     3541 aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat gattgagcta aacgatgatt
     3601 acacactgaa aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca gatgaaattg
     3661 aacgcgcgaa cgtctttaaa atgaacggca aatggtacct gttcactgac tcccgcggat
     3721 caaaaatgac gattgacggc attacgtcta acgatattta catgcttggt tatgtttcta
     3781 attctttaac tggcccatac aagccgctga acaaaactgg ccttgtgtta aaaatggatc
     3841 ttgatcctaa cgatgtaacc tttacttact cacacttcgc tgtacctcaa gcgaaaggaa
     3901 acaatgtcgt gattacaagc tatatgacaa acagaggatt ctacgcagac aaacaatcaa
     3961 cgtttgcgcc aagcttcctg ctgaacatca aaggcaagaa aacatctgtt gtcaaagaca
     4021 gcatccttga acaaggacaa ttaacagtta acaaataaca tatgcggtgt gaaataccgc
     4081 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     4141 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4201 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4261 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4321 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4381 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4441 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4501 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4561 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4621 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4681 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4741 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4801 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4861 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4921 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4981 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5041 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5101 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5161 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5221 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5281 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5341 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     5401 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5461 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5521 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5581 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5641 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     5701 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5761 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5821 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5881 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5941 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6001 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacct
//
