LOCUS       sequence-73667-e        8020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  loxP-kanMX-loxP cassette inserted into the ACT1 intron for PCR 
            amplification of the intron/kanMX module for one-step 
            homology-driven genomic disruptions .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8020)
  AUTHORS   Cheng TH, Chang CR, Joy P, Yablok S, Gartenberg MR
  TITLE     Controlling gene expression in yeast by inducible site-specific 
            recombination.
  JOURNAL   Nucleic Acids Res. 2000 Dec 15;28(24):E108.
  PUBMED    11121495
REFERENCE   2  (bases 1 to 8020)
  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: "Nucleic 
            Acids Res."; date: "2000-12-15"; volume: "28(24)"; pages: "E108"
COMMENT     Sequence Label: pRKO
FEATURES             Location/Qualifiers
     source          1..8020
                     /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"
     promoter        317..503
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             504..1163
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     rep_origin      complement(1423..1878)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1560..1581)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1772..1791
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2008..2030
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2022..2039
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2023..2039
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2049..2068
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2049..2067
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2096..2112
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2097..2113
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2177..2392
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     protein_bind    2907..2940
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            2995..4351
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2995..3338
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3339..4148
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3406..3425)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      4154..4351
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    4414..4447
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5193..5209)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5193..5209)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5246..5266)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5246..5264)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5285..5301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5285..5301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5298..5320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5309..5325
                     /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(5333..5339,5340..5357,5358..5363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5516..5533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5687..6275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5767..5786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6446..7237,7238..7306))
                     /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     7069..7088
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7307..7411)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7448..7951
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7993..8011
                     /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 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt
      241 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta
      301 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat
      361 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata
      421 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc
      481 aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa
      541 atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact
      601 cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga
      661 ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt
      721 ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca
      781 ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag
      841 taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag
      901 atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag
      961 atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta
     1021 ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca
     1081 aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct
     1141 ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgcagca tacgatatat
     1201 atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga acagtatgat
     1261 actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga ggcgcgcttt
     1321 ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc tatgcggtgt
     1381 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata
     1441 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     1501 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     1561 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     1621 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     1681 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     1741 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     1801 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     1861 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     1921 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     1981 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     2041 agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc ctcgatcgag
     2101 gtcgacggta tcgataagct tgatatcgaa ttcctgcagc ccgggggatc cggtgattga
     2161 ttgagcaagc tagcttttca attcatcatt ttttttttat tctttttttt gatttcggtt
     2221 tctttgaaat ttttttgatt cggtaatctc cgaacagaag gaagaacgaa ggaaggagca
     2281 cagacttaga ttggtatata tacgcatatg tagtgttgaa gaaacatgaa attgcccagt
     2341 attcttaacc caactgcaca gaacaaaaac ctgcaggaaa cgaagataaa tcatgtcgaa
     2401 agctacatat aaggaacgtg ctgctactca tcctagtcct gttgctgcca agctatttaa
     2461 tatcatgcac gaaaagcaaa caaacttgtg tgcttcattg gatgttcgta ccaccaagga
     2521 attactggag ttagttgaag cattaggtcc caaaatttgt ttactaaaaa cacatgtgga
     2581 tatcttgact gatttttcca tggagggcac agttaagccg ctaaaggcat tatccgccaa
     2641 gtacaatttt ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt
     2701 gcagtactct gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg
     2761 tgtggtgggc ccaggtattg ttagcggttt gaagcaggcg gcagaagaag taacaaagga
     2821 acctagaggg tatgttctag cgcttgcacc atcccattta actgtaagaa gaattgcacg
     2881 gtcccaattg ctcgacaacc cttaatataa cttcgtataa tgtatgctat acgaagttat
     2941 taggtctaga gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg
     3001 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac
     3061 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt
     3121 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga
     3181 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata
     3241 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta
     3301 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaggaa aagactcacg
     3361 tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
     3421 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
     3481 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
     3541 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
     3601 ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca ttccaggtat
     3661 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     3721 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
     3781 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     3841 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac
     3901 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     3961 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     4021 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     4081 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     4141 ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata
     4201 gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt
     4261 tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt
     4321 caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac gccgccatcc
     4381 agtgtcgaaa acgagctctc gagaaccctt aatataactt cgtataatgt atgctatacg
     4441 aagttattag gtgatatcag atccactagt ggcctatgcg tcgagagatt tctcttttac
     4501 ctttttttac tatttttcac tctcccataa cctcctatat tgactgatct gtaataacca
     4561 cgatattatt ggaataaata ggggcttgaa atttggaaaa aaaaaaaaac tgaaatattt
     4621 tcgtgataag tgatagtgat attcttcttt tatttgctac tgttactaag tctcatgtac
     4681 taacatcgat tgcttcattc tttttgttgc tatattatat gtttagcctt ttgatgttag
     4741 cagaattgtc atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca
     4801 ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa
     4861 gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag
     4921 acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacagga tctgacatta
     4981 ttattgttgg aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt
     5041 acagaaaagc aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg
     5101 tattataagt aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc
     5161 agttattacc cagcttgctc aatcaatcac cggatccact agttctagag cggccgccac
     5221 cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     5281 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatag
     5341 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa
     5401 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5461 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5521 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5581 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5641 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5701 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5761 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5821 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5881 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5941 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6001 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6061 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6121 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6181 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6241 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6301 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6361 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6421 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6481 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6541 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6601 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6661 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6721 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6781 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6841 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6901 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6961 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7021 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7081 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7141 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7201 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7261 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7321 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7381 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt
     7441 ccttttcatc acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa
     7501 attaaaaata gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat
     7561 gtaaaagact ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct
     7621 caggtattaa tgccgaattg tttcatcttg tctgtgtaga agaccacaca cgaaaatcct
     7681 gtgattttac attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg
     7741 tctaataaat atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt
     7801 tttttttctt cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac
     7861 aaaacataaa aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata
     7921 cgaggcgcgt gtaagttaca ggcaagcgat ccgtcctaag aaaccattat tatcatgaca
     7981 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
