LOCUS       sequence-449156-        6477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a color marker (uidA) cassette for disruption on KU70 gene
            in K. phaffii..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6477)
  AUTHORS   Strucko T, Gadar-Lopez AE, Frohling FB, Frost ET, Iversen EF, Olsson
            H, Jarczynska ZD, Mortensen UH
  TITLE     Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering 
            of Komagataella phaffii.
  JOURNAL   FEMS Yeast Res. 2024 Aug 23:foae026. doi: 10.1093/femsyr/foae026.
  PUBMED    39179418
REFERENCE   2  (bases 1 to 6477)
  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: "FEMS Yeast 
            Res. 2024 Aug 23:foae026. doi: 10.1093/femsyr/foae026."
COMMENT     Sequence Label: pDIV643
FEATURES             Location/Qualifiers
     source          1..6477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1131..1537)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     protein_bind    complement(2124..2157)
                     /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)"
     primer_bind     complement(2364..2383)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2389..2408)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2450..2541
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2542..2610,2611..3402)
                     /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     complement(2760..2779)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3524..4112
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4013..4032
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4266..4283
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4381..4398
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4382..4398
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4868..4901
                     /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)"
     terminator      complement(5571..5760)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
ORIGIN
        1 tattcccagc ttcaaaaccg attcctaagg ataaattgaa tcctacggcg gcggtctcgt
       61 ctatgactac gatcccatgc tcatctgccc aatcaagcat ctcttcggca tatgggtagt
      121 gactagttct gtaactgttg gcaccaatcc aatccatcaa agcatgatcg tgcaccataa
      181 gaacattgtc aaatccctta cctcgaaggt cagcatcctc atgtctccca aatcctgtga
      241 agtagaaagg tttgtggttt atcaaaaact gctccccctt cacggctacg gatctgatac
      301 cgactcgaag agggtagata tcacattctg tttgtgattt tgcggttaca cacaattcat
      361 aaaggtatcc ctcccctggt tgccataagt gtgggttgac gacttgaagt gtcccggagg
      421 taccctgacc ggttgccact acctgttggt cagcatcacg caactcgaca ctcacgtctc
      481 cgtttgcgac cacttgccaa tcaacagaag cgtgattgca gtcctgtgcc acatgggtga
      541 cgacggttat gtcgtccacc caggtgttag gagttgtata caacattact gaacgatgta
      601 tccctgcgta gttgaaaaag tcatggaaat aagactgctt ttttttacca ttctcgtcag
      661 ttatcaccat acctggtggg atagtctgcc agttaagttc gttgtttaca cagactgtga
      721 ttcgtactga tttcccagcg ataacgtatg gtgttacatc agcttcgaat ggagtatacc
      781 caccttggtg ctccataact tcctggttgt tcacccagac ctttccatag tgggtcactg
      841 catcgaaacg aagaacgatt cgttgacctg cccaaccttt agggataaaa acttctcgct
      901 ggtaccaaac attacctgcg tagttacgaa tatcagcatc ggcgaactga tcattgaatg
      961 aaccagggac ggcaatggct ctagactcct gcaaagcgga ctcccaccat cgctggtcaa
     1021 taccgcaatt ctctctgtca agactaaagg cccaaagacc gtcaagtttt ttgatctctc
     1081 ttgtaggggt ctctactggt cgcaacatat gacagatttt gtaattaaaa cttagattag
     1141 attgctatgc tttctttcta atgagcaaga agtaaaaaaa gttgtaatag aacaagaaaa
     1201 atgaaactga aacttgagaa attgaagacc gtttattaac ttaaatatca atgggaggtc
     1261 atcgaaagag aaaaaaatca aaaaaaaaaa ttttcaagaa aaagaaacgt gataaaaatt
     1321 tttattgcct ttttcgacga agaaaaagaa acgaggcggt ctcttttttc ttttccaaac
     1381 ctttagtacg ggtaattaac gacaccctag aggaagaaag aggggaaatt tagtatgctg
     1441 tgcttgggtg ttttgaagtg gtacggcgat gcgcggagtc cgagaaaatc tggaagagta
     1501 aaaaaggagt agaaacattt tgaagctatg gtgtgtgcgc aatgctttgg atgtcgtatt
     1561 gcttgctgac aacactcatc ttgcaatgct ttttattatt ctctgtgtta gggctaagtg
     1621 tgagaagaag agatgtgaaa gtcccccccc tcaaagctca ggcatgcgcg cttgcacgta
     1681 accccgtttc ctctataagt actaaagcct atacaaccct ttgcttcctc ctggcaaata
     1741 ctaggacgga gttccatatt actttcatgt tacccccctc aaagcagtcg ttcagttcgt
     1801 tgtaaaaatc atctaggata ccctcctttc ctcgttgctg cttccacgaa aacacagaag
     1861 gccaactaga tacaaaattg atgaaatcac tgattgtcat ctctttttcc aagtataaga
     1921 tatctccgtt tttgacccct tcctcttgtt taaaattaga aattttcact tcgtcaaaaa
     1981 gctcgctatc cagtaagatg tgattcaact cctttaaatg atttctgaaa tgtgtacggc
     2041 caggatccca atatgggccc aggtagtcag acccgtaggt gaaattaaac cctcagcgcg
     2101 gccgcattta aatccccact tcagaagttc ctatactttc tagagaatag gaacttcact
     2161 atagagtcga ataagggcga caccccataa ttagcccggg cgaaaggccc agtctttcga
     2221 ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcatgg ggagtcccca
     2281 cactaccatc ggcgctacgg cgtttcactt ctgagttcgg catggggtca ggtgggacca
     2341 ccgcgctact gccgccaggc aaacaagggg tgttatgagc catattcagg tataaatggg
     2401 ctcgcgataa tgttcagaat tggttaattg gttgtaacac tgacccctat ttgtttattt
     2461 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2521 taatattgaa aaaggaagaa tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2581 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2641 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2701 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2761 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     2821 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2881 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2941 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3001 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3061 gacgagcgtg acaccacgat gcctgtagcg atggcaacaa cgttgcgcaa actattaact
     3121 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3181 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatcc
     3241 ggagccggtg agcgtggttc tcgcggtatc atcgcagcgc tggggccaga tggtaagccc
     3301 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3361 cagatcgctg agataggtgc ctcactgatt aagcattggt aagcagagca ttacgctgac
     3421 ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgcgcgtcgt
     3481 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3541 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3601 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3661 caaatactgt tcttctagtg tagccgtagt tagcccacca cttcaagaac tctgtagcac
     3721 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3781 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3841 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3901 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3961 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4021 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4081 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4141 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4201 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4261 agcgcagcga gtcagtgagc gaggaagcgg aaggcgagag tagggaactg ccaggcatca
     4321 aactaagcag aaggcccctg acggatggcc tttttgcgtt tctacaaact ctttctgtgt
     4381 tgtaaaacga cggccagtct taagctcggg ccccctgggc ggttctgata acgagtaatc
     4441 gttaatccgc aaataacgta aaaacccgct tcggcgggtt tttttatggg gggagtttag
     4501 ggaaagagca tttgtcagaa tatttaaggg cgcctgtcac tttgcttgat atatgagaat
     4561 tatttaacct tataaatgag aaaaaagcaa cgcactttaa ataagatacg ttgctttttc
     4621 gattgatgaa cacctataat taaactattc atctattatt tatgattttt tgtatataca
     4681 atatttctag tttgttaaag agaattaaga aaataaatct cgaaaataat aaagggaaaa
     4741 tcagtttttg atatcaaaat tatacatgtc aacgataata caaaatataa tacaaactat
     4801 aagatgttat cagtatttat tatgcattta gaataaattt tgtgtcgccc ttattcgact
     4861 cactatagaa gttcctattc tctagaaagt ataggaactt cacttcattt tatttaaatt
     4921 tgcggccgcg ctgaggtctt aataggacag gtgaagtaaa cccgcttgat ctccttatgt
     4981 ctagaaattc gttggcggat ctcctcaatt gacatcgaac aagcatcttt atcatagaac
     5041 aaaatttctg agtattccgt cttgtcaaac ttgacacctg aaggcttgtt cagaagaaac
     5101 ggaatcaaag taatgtctac gtcattgtaa tcagccaggg ttttcttcaa gggaactctc
     5161 agctgagagt ttccattgta aggtttatcg ttattagtga acaagaaaat ctttttcaag
     5221 ttgtaatgtc ttccagtacg cttatgagtc ttcgccatgt aaaaaagaga ccttaagtga
     5281 aagaacagag tggctgataa cggtttactg tgttcaattg ggaattgctt ctcgagagga
     5341 ttaagcccac ttacatggtc ctccaagact tggtacaatt gtttcatcat ttccaaattc
     5401 aggtcttgta actcaaaaat ggggtaaatt tcgtcgtttt gaccaccgtc gtaattaata
     5461 aggtaacacc ctattcctgt accgggcaag gtaatgatta gctcagaaat aacctcactg
     5521 acattctcta agatgatctg gagctgagat ttcccttctg aagccgacac cttcgagcgt
     5581 cccaaaacct tctcaagcaa ggttttcagt ataatgttac atgcgtacac gcgtctgtac
     5641 agaaaaaaaa gaaaaatttg aaatataaat aacgttctta atactaacat aactataaaa
     5701 aaataaatag ggacctagac ttcaggttgt ctaactcctt ccttttcggt tagagcggat
     5761 gaatgcacgc gattcattgt ttacctccct gctgaggctt ctctccaaaa ttcatcccgg
     5821 tccaacgctt ctgaagtaag aaagcggcag atttaggctt tcgatctctt gtgaatatac
     5881 cctttttgtt acctcctaca cgcaatatac cttgtgatgt ggcgaaatcg gcgaaattcc
     5941 acacctgctc acctacaact gcagaaactc tgtcaaacac acggtgatac atgtctaacc
     6001 aggcacactg gtactcttca ctccacatgt cagtatacat actatgcaat cctgcaaggg
     6061 tgtccacccc atactcggta ataataattg gttggtgaag tttttcctgc caagcaagaa
     6121 gctctttctc taagactttt tctgctgtct ctaagtctcc tgattgcaca taccacccat
     6181 agtaacggtt aaggcacaac acgtcaaaaa ggtcggaaat tgtgtcagtg tgggcgtcgc
     6241 aaaacatgac gtttacgcat gtaattggtc gggttggatc taatttacgt gtagcttctg
     6301 caagtggggc gaagtattca cgggctcctt gaggtcgtgt gtctggctca ttggcaatgg
     6361 accacataac aacggatggg tggtttttat ctcttgcgat caactcctta atggcctgaa
     6421 ggtgtgcctg ttgggtctct ccgtttacag cctcctcgga gtaaagttcc ttaggct
//
