LOCUS       sequence-212774-        2642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for assembly of donor DNA templates consisting of two 
            expression units.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2642)
  AUTHORS   Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer R
  TITLE     CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia 
            pastoris.
  JOURNAL   Methods Mol Biol. 2019;1923:211-225. doi: 
            10.1007/978-1-4939-9024-5_9.
  PUBMED    30737742
REFERENCE   2  (bases 1 to 2642)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "1923:211-225. doi"; pages: " 
            10.1007/978-1-4939-9024-5_9"
COMMENT     Sequence Label: BB3nK_ext_AC
FEATURES             Location/Qualifiers
     source          1..2642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     gene            785..2141
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        785..1128
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1129..1938
                     /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(1196..1215)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1944..2141
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 cgctgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtgg agcgcaaaaa accccgcccc tgacagggcg gggttttttc
      721 gcgatcgaag acccaattgg agaccctcga gggatccggt ctccgatcgg gtcttcaatt
      781 catggacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc
      841 atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc
      901 catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacgtg
      961 cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta
     1021 gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa
     1081 aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaggaa
     1141 aagactcacg tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     1201 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     1261 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     1321 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     1381 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca
     1441 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     1501 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     1561 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     1621 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg
     1681 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     1741 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     1801 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     1861 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     1921 ctcgatgagt ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt
     1981 catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt
     2041 atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa
     2101 tatcatgcgt caatcgtatg tgaatgctgg tcgctatact ggcttgtaag caatctggac
     2161 aattatgtaa gcgggttacg taaacagtta tgtaagcaga aaaatttcaa acgacaaaac
     2221 ttggggtcta cagacacagt agccagaaga ttgcactacc attcgactcc tcatgaccca
     2281 ctctttcgat ccatgtagtt aggttaccgt ttttcctaat atttaaggat gttgaaaatt
     2341 cattttcatt tttttcgttt ttaagatttt ctcacaactc ttccaaagat tactagttga
     2401 cttttcaaat atttagggta tttttctcac tttttcctag caaactccaa ttggtgggtt
     2461 cagtgcaatg gagtatcacc ttgcaaccac aacgtaatag ctaacttgtg gccaccatgt
     2521 ctggttgtag agataattgg attctaatgt ggatcacatg actactcacg tgtcaaaaac
     2581 ccaacctgac ttggcccagc ttagcaagaa tatttcgaat ccactcttgt ggcctagtgg
     2641 ac
//
