LOCUS       sequence-468388-        4583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            GFP and selection on Nat..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4583)
  AUTHORS   Colarusso AV, Williams AM, Gladfelter AS, Wirshing ACE, Lew DJ
  TITLE     Optimized vectors for genetic engineering of Aureobasidium 
            pullulans.
  JOURNAL   Mol Biol Cell. 2025 Jun 1;36(6):mr5. doi: 10.1091/mbc.E25-02-0059. 
            Epub 2025 Apr 9.
  PUBMED    40202829
REFERENCE   2  (bases 1 to 4583)
  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: "Mol Biol 
            Cell."; date: "2025-06-1"; volume: "36(6):mr5. doi"; pages: " 
            10.1091/mbc.E25-02-0059. Epub 2025 Apr 9"
COMMENT     Sequence Label: pAPInt-GFP-Nat
FEATURES             Location/Qualifiers
     source          1..4583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..36)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             553..1125
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1131..1328
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(1371..1390)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1372..1390)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1477..1494)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1648..2236)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1728..1747)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2407..3198,3199..3267))
                     /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     3030..3049
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3268..3372)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3440..3458
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3496..3518)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3618..3637
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3718..3736
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3718..3735
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4109..4128)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     terminator      join(4540..4583,1..144)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
ORIGIN
        1 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
       61 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc
      121 tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtgg tgcgggtgaa
      181 aggtcttggt caagtcgcaa agtgtttgtg aaaaagacgt agcgggggaa gcgcacacac
      241 gcccgccttt tatagacgaa gcttcgagca ggtcatttaa cgatgcttag tagcaatata
      301 caataaagcc taattgggac taattacggt cacgtgaaaa actcaccatg tagcctttta
      361 tggaaatatt gtacacgcta ctaagccact gtaatatcgg cctcatcgtc tccgcttgga
      421 gcataaaagg cgacgctcct cgccattccc ccaagtcgtt ttccgtattc tgctacgctt
      481 caagctttga cgtttctctt caagactcac tcacgaccct ctctctcacg cacaccttac
      541 acagcagaat tcatgggtac cactcttgac gacacggctt accggtaccg caccagtgtc
      601 ccgggggacg ccgaggccat cgaggcactg gatgggtcct tcaccaccga caccgtcttc
      661 cgcgtcaccg ccaccgggga cggcttcacc ctgcgggagg tgccggtgga cccgcccctg
      721 accaaggtgt tccccgacga cgaatcggac gacgaatcgg acgacgggga ggacggcgac
      781 ccggactccc ggacgttcgt cgcgtacggg gacgacggcg acctggcggg cttcgtggtc
      841 gtctcgtact ccggctggaa ccgccggctg accgtcgagg acatcgaggt cgccccggag
      901 caccgggggc acggggtcgg gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag
      961 cggggcgccg ggcacctctg gctggaggtc accaacgtca acgcaccggc gatccacgcg
     1021 taccggcgga tggggttcac cctctgcggc ctggacaccg ccctgtacga cggcaccgcc
     1081 tcggacggcg agcaggcgct ctacatgagc atgccctgcc cctaatcagt actgacaata
     1141 aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc
     1201 tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag
     1261 atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg
     1321 ctatactgca gatccactag tggcctatgc ggccgcggat ctgccggtct ccctatagtg
     1381 agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg
     1441 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     1501 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     1561 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     1621 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     1681 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     1741 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     1801 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     1861 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     1921 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     1981 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2041 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     2101 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2161 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2221 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2281 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     2341 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2401 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     2461 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     2521 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     2581 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     2641 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     2701 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     2761 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     2821 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     2881 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     2941 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3001 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3061 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3121 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3181 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3241 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     3301 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3361 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     3421 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     3481 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     3541 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     3601 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat
     3661 attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt
     3721 taggtgacac tatagaacgc ggccgccagc tgaagcttcg tacgctgcag gtcgacggat
     3781 cggtgacggt gctggttctg gttctatgtc taagggcgag gagctcttca ccggcgtcgt
     3841 ccctatcctc gtcgagctcg acggcgacgt caacggccac aagttctctg tctctggcga
     3901 gggcgagggc gacgccacct acggcaagct caccctcaag ttcatctgca ccaccggcaa
     3961 gctccctgtc ccttggccta ccctcgtcac caccttcggc tacggcgtcc aatgcttcgc
     4021 ccgctaccct gaccacatga agcaacacga cttcttcaag tctgccatgc ctgagggcta
     4081 cgtccaagag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     4141 caagttcgag ggcgacaccc tcgtcaaccg catcgagctc aagggcatcg acttcaagga
     4201 ggacggcaac atcctcggcc acaagctcga gtacaactac aactctcaca acgtctacat
     4261 catggccgac aagcaaaaga acggcatcaa ggtcaacttc aagatccgcc acaacatcga
     4321 ggacggctct gtccaactcg ccgaccacta ccaacaaaac acccctatcg gcgacggccc
     4381 tgtcctcctc cctgacaacc actacctctc tacccaatct gccctctcta aggaccctaa
     4441 cgagaagcgc gaccacatgg tcctcctcga gttcgtcacc gccgccggca tcacccacgg
     4501 catggacgag ctctacaagt gagcgccact tctaaataag cgaatttctt atgatttatg
     4561 atttttatta ttaaataagt tat
//
