LOCUS       sequence-136113-        6968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains the CORE cassette with convergent KlURA3 and KanMX4 markers
            and the I-SceI gene under the inducible GAL1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6968)
  AUTHORS   Storici F, Durham CL, Gordenin DA, Resnick MA
  TITLE     Chromosomal site-specific double-strand breaks are efficiently 
            targeted for repair by oligonucleotides in yeast.
  JOURNAL   Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14994-9. Epub 2003 Nov 
            20.
  PUBMED    14630945
REFERENCE   2  (bases 1 to 6968)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2003-12-9"; volume: "100(25)"; pages: 
            "14994-9. Epub 2003 Nov 20"
COMMENT     Sequence Label: pGSKU
FEATURES             Location/Qualifiers
     source          1..6968
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(138..143,144..161,162..168)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    176..192
                     /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)."
     primer_bind     181..203
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     200..216
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     200..216
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        237..255
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     gene            complement(1843..3199)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(1843..2040)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2046..2855)
                     /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     2769..2788
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2856..3199)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(3981..4422)
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(3987..4010)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    4305..4422
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     complement(4711..4728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4882..5470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4962..4981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5641..6432,6433..6501))
                     /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     6264..6283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6502..6606)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6674..6692
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6730..6752)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6852..6871
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6952..6968,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6952..6968,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc ctggcacgac
       61 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
      121 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
      181 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcggaatt
      241 aaccctcact aaagggaaca aaagctggta ccgggcccgg agacaatcat atgggagaag
      301 caattggaag atagaaaaaa ggtactcggt acataaatat atgtgattct gggtagaaga
      361 tcggtctgca ttggatggtg gtaacgcatt tttttacaca cattacttgc ctcgagcatc
      421 aaatggtggt tattcgtgga tctatatcac gtgatttgct taagaattgt cgttcatggt
      481 gacactttta gctttgacat gattaagctc atctcaattg atgttatcta aagtcatttc
      541 aactatctaa gatgtggttg tgattgggcc attttgtgaa agccagtacg ccagcgtcaa
      601 tacactcccg tcaattagtt gcaccatgtc cacaaaatca tataccagta gagctgagac
      661 tcatgcaagt ccggttgcat cgaaactttt acgtttaatg gatgaaaaga agaccaattt
      721 gtgtgcttct cttgacgttc gttcgactga tgagctattg aaactagttg aaacgttggg
      781 tccatacatt tgccttttga aaacacacgt tgatatcttg gatgatttca gttatgaggg
      841 tactgtcgtt ccattgaaag cattggcaga gaaatacaag ttcttgatat ttgaggacag
      901 aaaattcgcc gatatcggta acacagtcaa attacaatat acatcgggcg tttaccgtat
      961 cgcagaatgg tctgatatca ccaacgccca cggggttact ggtgctggta ttgttgctgg
     1021 cttgaaacaa ggtgcgcaag aggtcaccaa agaaccaagg ggattattga tgcttgctga
     1081 attgtcttcc aagggttctc tagcacacgg tgaatatact aagggtaccg ttgatattgc
     1141 aaagagtgat aaagatttcg ttattgggtt cattgctcag aacgatatgg gaggaagaga
     1201 agaagggttt gattggctaa tcatgacccc aggtgtaggt ttagacgaca aaggcgatgc
     1261 attgggtcag cagtacagaa ccgtcgacga agttgtaagt ggtggatcag atatcatcat
     1321 tgttggcaga ggacttttcg ccaagggtag agatcctaag gttgaaggtg aaagatacag
     1381 aaatgctgga tgggaagcgt accaaaagag aatcagcgct ccccattaat tatacaggaa
     1441 acttaataga acaaatcaca tatttaatct aatagccacc tgcattggca cggtgcaaca
     1501 ctcacttcaa cttcatctta caaaagatca cgtgatctgt tgtattgaac tgaaaatttt
     1561 ttgtttgctt ctctctctct ctttcattat gtgagagttt aaaaaccaga aactacatca
     1621 tcgaaaaaga gtttaaacca ttacaaccat tgcgataagc cctctcaaac ttcctccaga
     1681 ctgtttgtca tccaattggt aagattaatt atcatatacc ccggataaga aaagaagcaa
     1741 gcacacaatg gatcaactta atggtaagga acaacaagcg gccgccaccg cggtggagct
     1801 cgttttcgac actggatggc ggcgttagta tcgaatcgac agcagtatag cgaccagcat
     1861 tcacatacga ttgacgcatg atattacttt ctgcgcactt aacttcgcat ctgggcagat
     1921 gatgtcgagg cgaaaaaaaa tataaatcac gctaacattt gattaaaata gaacaactac
     1981 aatataaaaa aactatacaa atgacaagtt cttgaaaaca agaatctttt tattgtcagt
     2041 actgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2101 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2161 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2221 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2281 acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca
     2341 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2401 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2461 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2521 ggatattctt ctaatacctg gaatgctgtt ttgccgggga tcgcagtggt gagtaaccat
     2581 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2641 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2701 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2761 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2821 cgcggcctcg aaacgtgagt cttttcctta cccatggttg tttatgttcg gatgtgatgt
     2881 gagaactgta tcctagcaag attttaaaag gaagtatatg aaagaagaac ctcagtggca
     2941 aatcctaacc ttttatattt ctctacaggg gcgcggcgtg gggacaattc aacgcgtctg
     3001 tgaggggagc gtttccctgc tcgcaggtct gcagcgagga gccgtaattt ttgcttcgcg
     3061 ccgtgcggcc atcaaaatgt atggatgcaa atgattatac atggggatgt atgggctaaa
     3121 tgtacgggcg acagtcacat catgcccctg agctgcgcac gtcaagactg tcaaggaggg
     3181 tattctgggc ctccatgtcg ctggccgggt gacccggcgg ggacgaggca agctaaacag
     3241 atctgactta ttatttcagg aaagtttcgg aggagatagt gttcggcagt ttgtacatca
     3301 tctgcgggat caggtacggt ttgatcaggt tgtagaagat caggtaagac atagaatcga
     3361 tgtagatgat cggtttgttt ttgttgattt ttacgtaaca gttcagttgg aatttgttac
     3421 gcagaccctt aaccaggtat tctacttctt cgaaagtgaa agactgggtg ttcagtacga
     3481 tcgatttgtt ggtagagttt ttgttgtaat cccatttacc accatcatcc atgaaccagt
     3541 atgccagaga catcggggtc aggtagtttt caaccaggtt gttcgggatg gtttttttgt
     3601 tgttaacgat gaacaggtta gccagtttgt tgaaagcttg gtgtttgaaa gtctgggcgc
     3661 cccaggtgat taccaggtta cccaggtggt taacacgttc ttttttgtgc ggcggggaca
     3721 gtacccactg atcgtacagc agacatacgt ggtccatgta tgctttgttt ttccactcga
     3781 actgcataca gtaggtttta ccttcatcac gagaacggat gtaagcatca cccaggatca
     3841 gaccgatacc tgcttcgaac tgttcgatgt tcagttcgat cagctgggat ttgtattctt
     3901 tcagcagttt agagttcgga cccaggttca ttacctggtt ttttttgatg tttttcatat
     3961 gcatggatcc ggggtttttt ctccttgacg ttaaagtata gaggtatatt aacaattttt
     4021 tgttgatact tttattacat ttgaataaga agtaatacaa accgaaaatg ttgaaagtat
     4081 tagttaaagt ggttatgcag tttttgcatt tatatatctg ttaatagatc aaaaatcatc
     4141 gcttcgctga ttaattaccc cagaaataag gctaaaaaac taatcgcatt atcatcctat
     4201 ggttgttaat ttgattcgtt catttgaagg tttgtggggc caggttactg ccaatttttc
     4261 ctcttcataa ccataaaagc tagtattgta gaatctttat tgttcggagc agtgcggcgc
     4321 gaggcacatc tgcgtttcag gaacgcgacc ggtgaagacg aggacgcacg gaggagagtc
     4381 ttccttcgga gggctgtcac ccgctcggcg gcttctaatc cgtacttcaa tatagcaatg
     4441 agcagttaag cgtattactg aaagttccaa agagaaggtt tttttaggct aagataatgg
     4501 ggctctttac atttccacaa catataagta agattagata tggatatgta tatggatatg
     4561 tatatggtgg taatgccatg taatatgatt attaaacttc tttgcgtcca tccaacgaga
     4621 tctggcgcgc cttaattaac ccaacctgca ttaatgaatc ggccaacgcg cggggagagg
     4681 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4741 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4801 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4861 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4921 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4981 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5041 cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag
     5101 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5161 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5221 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5281 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     5341 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5401 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5461 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5521 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5581 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5641 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5701 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5761 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5821 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5881 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5941 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6001 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6061 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6121 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6181 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6241 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6301 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6361 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6421 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6481 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6541 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6601 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     6661 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     6721 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     6781 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     6841 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg acatattgtc
     6901 gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg
     6961 acactata
//
