LOCUS       sequence-109912-        3939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  source of KmR FRT cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3939)
  AUTHORS   Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer 
            RR, Schweizer HP
  TITLE     A Tn7-based broad-range bacterial cloning and expression system.
  JOURNAL   Nat Methods. 2005 Jun;2(6):443-8.
  PUBMED    15908923
REFERENCE   2  (bases 1 to 3939)
  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: "Nat 
            Methods."; date: "2005-06"; volume: "2(6)"; pages: "443-8"
COMMENT     Sequence Label: pFKM1
FEATURES             Location/Qualifiers
     source          1..3939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    464..511
                     /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)."
     CDS             complement(620..1435)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(620..1435)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1427..1446
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1585..1632
                     /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(1718..1734)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1718..1734)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1731..1753)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1742..1758
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1766..1772,1773..1790,1791..1796))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1811..1832
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1949..1966)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2120..2708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2200..2219)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2879..3670,3671..3739))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3502..3521
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3740..3844)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3912..3930
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg
      421 actctagagg atccccgggt accgagctcg aattggggat cttgaagtac ctattccgaa
      481 gttcctattc tctagaaagt ataggaactt cagagcgctt ttgaagctga tgtgcttaaa
      541 aacttactca atggaataat tctagatggg aagtcagcgt aatgctctgc cagtgttaca
      601 accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
      661 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      721 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      781 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      841 aatcaccatg agtgacgact gaatccggtg agaatggcaa gagcttatgc atttctttcc
      901 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      961 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     1021 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1081 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttgccg gggatcgcag
     1141 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1201 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1261 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1321 tcgcacctga ttgcccgaca ttatcgcgtg cccatttata cccatataaa tcagcatcca
     1381 tgttggaatt taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac
     1441 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     1501 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttcccat ccattgctgt
     1561 tgacaaaggg aatcagggga tcttgaagtt cctattccga agttcctatt ctctagaaag
     1621 tataggaact tcagagcgct tttgaagcta attcgagctc ggtacccggg gatcctctag
     1681 agtcgacctg caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     1741 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1801 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1861 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1921 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1981 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2041 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2101 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2161 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2221 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2281 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2341 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2401 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2461 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2521 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     2581 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2641 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2701 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2761 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2821 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2881 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2941 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3001 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3061 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3121 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3181 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3241 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3301 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3361 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3421 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3481 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3541 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3601 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3661 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3721 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3781 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3841 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     3901 taacctataa aaataggcgt atcacgaggc cctttcgtc
//
