LOCUS       sequence-469196-        4517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            mStayGold and selection on Nat..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4517)
  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 4517)
  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-Nat
FEATURES             Location/Qualifiers
     source          1..4517
                     /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..2133
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      2139..2336
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2379..2398)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2380..2398)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2485..2502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2656..3244)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2736..2755)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3415..4206,4207..4275))
                     /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     4038..4057
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4276..4380)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4448..4466
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(4504..4517,1..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 atgggtacca ctcttgacga cacggcttac cggtaccgca ccagtgtccc gggggacgcc
     1621 gaggccatcg aggcactgga tgggtccttc accaccgaca ccgtcttccg cgtcaccgcc
     1681 accggggacg gcttcaccct gcgggaggtg ccggtggacc cgcccctgac caaggtgttc
     1741 cccgacgacg aatcggacga cgaatcggac gacggggagg acggcgaccc ggactcccgg
     1801 acgttcgtcg cgtacgggga cgacggcgac ctggcgggct tcgtggtcgt ctcgtactcc
     1861 ggctggaacc gccggctgac cgtcgaggac atcgaggtcg ccccggagca ccgggggcac
     1921 ggggtcgggc gcgcgttgat ggggctcgcg acggagttcg cccgcgagcg gggcgccggg
     1981 cacctctggc tggaggtcac caacgtcaac gcaccggcga tccacgcgta ccggcggatg
     2041 gggttcaccc tctgcggcct ggacaccgcc ctgtacgacg gcaccgcctc ggacggcgag
     2101 caggcgctct acatgagcat gccctgcccc taatcagtac tgacaataaa aagattcttg
     2161 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa
     2221 atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa
     2281 gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgcaga
     2341 tccactagtg gcctatgcgg ccgcggatct gccggtctcc ctatagtgag tcgtattaat
     2401 ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     2461 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     2521 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2581 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2641 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2701 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2761 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2821 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2881 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2941 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3001 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3061 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3121 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3181 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3241 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3301 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     3361 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     3421 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3481 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     3541 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     3601 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     3661 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     3721 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     3781 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     3841 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     3901 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     3961 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4021 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4081 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4141 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4201 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4261 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4321 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     4381 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     4441 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     4501 aaacctctga cacatgc
//
