LOCUS       sequence-468367-        5039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            GFP and selection on Hyg..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5039)
  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 5039)
  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-Hyg
FEATURES             Location/Qualifiers
     source          1..5039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..36)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             553..1581
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             553..1581
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      1587..1784
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(1827..1846)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1828..1846)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1933..1950)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2104..2692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2184..2203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2863..3654,3655..3723))
                     /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     3486..3505
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3724..3828)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3896..3914
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3952..3974)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4074..4093
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4174..4192
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4174..4191
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4565..4584)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     terminator      join(4996..5039,1..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 tcatgggtaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
      601 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
      661 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
      721 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
      781 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
      841 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
      901 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
      961 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     1021 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     1081 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     1141 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     1201 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     1261 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     1321 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     1381 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     1441 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     1501 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     1561 cgtccgaggg caaaggaata atcagtactg acaataaaaa gattcttgtt ttcaagaact
     1621 tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga
     1681 tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag
     1741 taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgcagatc cactagtggc
     1801 ctatgcggcc gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca
     1861 ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1921 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     1981 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2041 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2101 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2161 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2221 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2281 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2341 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2401 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2461 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2521 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2581 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2641 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2701 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2761 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2821 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     2881 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     2941 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3001 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3061 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3121 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3181 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3241 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3301 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3361 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3421 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3481 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3541 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3601 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3661 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     3721 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     3781 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     3841 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     3901 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     3961 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     4021 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     4081 agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa
     4141 ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaacgcggcc
     4201 gccagctgaa gcttcgtacg ctgcaggtcg acggatcggt gacggtgctg gttctggttc
     4261 tatgtctaag ggcgaggagc tcttcaccgg cgtcgtccct atcctcgtcg agctcgacgg
     4321 cgacgtcaac ggccacaagt tctctgtctc tggcgagggc gagggcgacg ccacctacgg
     4381 caagctcacc ctcaagttca tctgcaccac cggcaagctc cctgtccctt ggcctaccct
     4441 cgtcaccacc ttcggctacg gcgtccaatg cttcgcccgc taccctgacc acatgaagca
     4501 acacgacttc ttcaagtctg ccatgcctga gggctacgtc caagagcgca ccatcttctt
     4561 caaggacgac ggcaactaca agacccgcgc cgaggtcaag ttcgagggcg acaccctcgt
     4621 caaccgcatc gagctcaagg gcatcgactt caaggaggac ggcaacatcc tcggccacaa
     4681 gctcgagtac aactacaact ctcacaacgt ctacatcatg gccgacaagc aaaagaacgg
     4741 catcaaggtc aacttcaaga tccgccacaa catcgaggac ggctctgtcc aactcgccga
     4801 ccactaccaa caaaacaccc ctatcggcga cggccctgtc ctcctccctg acaaccacta
     4861 cctctctacc caatctgccc tctctaagga ccctaacgag aagcgcgacc acatggtcct
     4921 cctcgagttc gtcaccgccg ccggcatcac ccacggcatg gacgagctct acaagtgagc
     4981 gccacttcta aataagcgaa tttcttatga tttatgattt ttattattaa ataagttat
//
