LOCUS       sequence-469195-        4973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            mStayGold and selection on Hyg..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4973)
  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 4973)
  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-mStayGold-Hyg
FEATURES             Location/Qualifiers
     source          1..4973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     109..128
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        209..227
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     209..226
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             297..944
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ivorra-Molla et al., 2023)"
                     /label=mStayGold (E138D)
                     /note="contains the monomerizing E138D mutation"
                     /translation="ASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKLP
                     MSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFMK
                     NGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKSK
                     CVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     terminator      965..1152
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1020..1044)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             1561..2589
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1561..2589
                     /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      2595..2792
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2835..2854)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2836..2854)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2941..2958)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3112..3700)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3192..3211)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3871..4662,4663..4731))
                     /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     4494..4513
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4732..4836)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4904..4922
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(4960..4973,1..9))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
       61 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
      121 agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat
      181 acataacctt atgtatcata cacatacgat ttaggtgaca ctatagaacg cggccgccag
      241 ctgaagcttc gtacgctgca ggtcgacgga tcggtgacgg tgctggttct ggttctgcct
      301 ctaccccttt caagttccaa ctcaagggca ccatcaacgg caagtctttc accgtcgagg
      361 gcgagggcga gggcaactct cacgagggct ctcacaaggg caagtacgtc tgcacctctg
      421 gcaagctccc tatgtcttgg gccgccctcg gcacctcttt cggctacggc atgaagtact
      481 acaccaagta cccttctggc ctcaagaact ggttccacga ggtcatgcct gagggcttca
      541 cctacgaccg ccacatccaa tacaagggcg acggctctat ccacgccaag caccaacact
      601 tcatgaagaa cggcacctac cacaacatcg tcgagttcac cggccaagac ttcaaggaga
      661 actctcctgt cctcaccggc gacatgaacg tctctctccc taacgacgtc caacacatcc
      721 ctcgcgacga cggcgtcgag tgccctgtca ccctcctcta ccctctcctc tctgacaagt
      781 ctaagtgcgt cgaggcccac caaaacacca tctgcaagcc tctccacaac caacctgccc
      841 ctgacgtccc ttaccactgg atccgcaagc aatacaccca atctaaggac gacaccgagg
      901 agcgcgacca catctgccaa tctgagaccc tcgaggccca cctctgagcg ccacttctaa
      961 ataagcgaat ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag
     1021 tgtatacaaa ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa
     1081 ctctttcctg taggtcaggt tgctttctca ggtatagtat gaggtcgctc ttattgacca
     1141 cacctctacc ggcagatccg ctagggataa cagggtggtg cgggtgaaag gtcttggtca
     1201 agtcgcaaag tgtttgtgaa aaagacgtag cgggggaagc gcacacacgc ccgcctttta
     1261 tagacgaagc ttcgagcagg tcatttaacg atgcttagta gcaatataca ataaagccta
     1321 attgggacta attacggtca cgtgaaaaac tcaccatgta gccttttatg gaaatattgt
     1381 acacgctact aagccactgt aatatcggcc tcatcgtctc cgcttggagc ataaaaggcg
     1441 acgctcctcg ccattccccc aagtcgtttt ccgtattctg ctacgcttca agctttgacg
     1501 tttctcttca agactcactc acgaccctct ctctcacgca caccttacac agcagaattc
     1561 atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc
     1621 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     1681 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     1741 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     1801 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     1861 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     1921 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     1981 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     2041 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     2101 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     2161 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     2221 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     2281 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     2341 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     2401 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     2461 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     2521 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     2581 aaggaataat cagtactgac aataaaaaga ttcttgtttt caagaacttg tcatttgtat
     2641 agttttttta tattgtagtt gttctatttt aatcaaatgt tagcgtgatt tatatttttt
     2701 ttcgcctcga catcatctgc ccagatgcga agttaagtgc gcagaaagta atatcatgcg
     2761 tcaatcgtat gtgaatgctg gtcgctatac tgcagatcca ctagtggcct atgcggccgc
     2821 ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg ttaacctgca
     2881 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2941 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3001 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3061 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3121 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3181 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3241 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3301 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3361 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3421 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3481 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3541 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3601 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3661 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3721 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3781 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3841 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3901 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3961 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4021 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4081 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4141 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4201 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4261 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4321 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4381 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4441 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4501 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4561 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4621 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4681 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4741 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4801 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4861 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     4921 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgc
//
