LOCUS       sequence-128203-        5846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid - kanamycin resistant donor cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5846)
  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 5846)
  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-neo
FEATURES             Location/Qualifiers
     source          1..5846
                     /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..1577
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(837..856)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1447..1466
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    1608..1641
                     /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             1922..2113
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2107..2129)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2270..2289
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             join(2436..2522,2523..3857)
                     /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(3926..3943)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4097..4685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4177..4196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4856..5647,5648..5716))
                     /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     5479..5498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5717..5821)
                     /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 accatgggat
      781 cggccattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
      841 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
      901 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
      961 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     1021 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     1081 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     1141 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     1201 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     1261 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     1321 acggcgatga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     1381 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     1441 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     1501 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     1561 ttgacgagtt cttctgaggg gatcaattct ctagagctcg ctgatcagaa gttcctattc
     1621 tctagaaagt ataggaactt cgatggcgcc tcatccctga agccaatagg gataacaggg
     1681 taatgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     1741 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     1801 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     1861 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     1921 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     1981 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     2041 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     2101 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     2161 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     2221 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     2281 cagattgtac tgagagtgca ccatatgtac tttggcgtca ccccttacat attttaggtc
     2341 tttttttatt gtgcgtaact aacttgccat cttcaaacag gagggctgga agaagcagac
     2401 cgctaacaca gtacataaaa aaggagacat gaacgatgaa catcaaaaag tttgcaaaac
     2461 aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact caagcgtttg
     2521 cgaaagaaac gaaccaaaag ccatataagg aaacatacgg catttcccat attacacgcc
     2581 atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa atatcaagtt cctgaattcg
     2641 attcgtccac aattaaaaat atctcttctg caaaaggcct ggacgtttgg gacagctggc
     2701 cattacaaaa cgctgacggc actgtcgcaa actatcacgg ctaccacatc gtctttgcat
     2761 tagccggaga tcctaaaaat gcggatgaca catcgattta catgttctat caaaaagtcg
     2821 gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat
     2881 tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga atggtcaggt tcagccacat
     2941 ttacatctga cggaaaaatc cgtttattct acactgattt ctccggtaaa cattacggca
     3001 aacaaacact gacaactgca caagttaacg tatcagcatc agacagctct ttgaacatca
     3061 acggtgtaga ggattataaa tcaatctttg acggtgacgg aaaaacgtat caaaatgtac
     3121 agcagttcat cgatgaaggc aactacagct caggcgacaa ccatacgctg agagatcctc
     3181 actacgtaga agataaaggc cacaaatact tagtatttga agcaaacact ggaactgaag
     3241 atggctacca aggcgaagaa tctttattta acaaagcata ctatggcaaa agcacatcat
     3301 tcttccgtca agaaagtcaa aaacttctgc aaagcgataa aaaacgcacg gctgagttag
     3361 caaacggcgc tctcggtatg attgagctaa acgatgatta cacactgaaa aaagtgatga
     3421 aaccgctgat tgcatctaac acagtaacag atgaaattga acgcgcgaac gtctttaaaa
     3481 tgaacggcaa atggtacctg ttcactgact cccgcggatc aaaaatgacg attgacggca
     3541 ttacgtctaa cgatatttac atgcttggtt atgtttctaa ttctttaact ggcccataca
     3601 agccgctgaa caaaactggc cttgtgttaa aaatggatct tgatcctaac gatgtaacct
     3661 ttacttactc acacttcgct gtacctcaag cgaaaggaaa caatgtcgtg attacaagct
     3721 atatgacaaa cagaggattc tacgcagaca aacaatcaac gtttgcgcca agcttcctgc
     3781 tgaacatcaa aggcaagaaa acatctgttg tcaaagacag catccttgaa caaggacaat
     3841 taacagttaa caaataacat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     3901 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3961 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4021 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4081 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4141 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4201 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4261 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4321 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4381 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4441 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4501 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     4561 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4621 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4681 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4741 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4801 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4861 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4921 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4981 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5041 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5101 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5161 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5221 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5281 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5341 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5401 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5461 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5521 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5581 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5641 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5701 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5761 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5821 gcacatttcc ccgaaaagtg ccacct
//
