LOCUS       sequence-468406-        4754 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            mStayGold and selection on G418..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4754)
  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 4754)
  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-G418
FEATURES             Location/Qualifiers
     source          1..4754
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     847..866
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        847..865
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(909..1106)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     terminator      complement(2330..2517)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2438..2462
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2538..3185)
                     /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"
     promoter        complement(3255..3273)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3256..3273)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3354..3373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3473..3495
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3533..3551)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3619..3723
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3724..3792,3793..4584)
                     /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     complement(3942..3961)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggttaa cctggcttat
      841 cgaaattaat acgactcact atagggagac cggcagatcc gcggccgcat aggccactag
      901 tggatctgca gtatagcgac cagcattcac atacgattga cgcatgatat tactttctgc
      961 gcacttaact tcgcatctgg gcagatgatg tcgaggcgaa aaaaaatata aatcacgcta
     1021 acatttgatt aaaatagaac aactacaata taaaaaaact atacaaatga caagttcttg
     1081 aaaacaagaa tctttttatt gtcagtactg actagaagaa ctcgtcgagc atgaggtgga
     1141 actggagctt gttcatgtcc gggttgtcga tgccgtactt ctggaagagg cgcttctgga
     1201 gcgacggcga gaactcgccg aggcagttcc agaggatggc gaggtcctgg tagcggtcgg
     1261 cgatgccgac gcggccgacg tcgatgcagc cgatgagctt gccctcgtcg aagatgaggt
     1321 tgtcgagcga gaagtcgccg tgcgtgacga ccgagtccgg cgagaacggg aggagcttgt
     1381 gcatctcctt ccagacctgc tcgaccggcc agccgttgcg ctcgtcgtcg aagtccgagg
     1441 cgtcgacgag gccgttgttc atgcgcgact gggcctgggc gaggcggaag acgcggtccg
     1501 agttgaacgg gcagttgcag accgggatcg agtggaggcg gcggaggaag acggcgaggg
     1561 cgtcgacgat gttctcgccc gagtccgggt actcctcgag gacctggaag gccgtcttgc
     1621 ccgggatggc cgtcgtgagg agccaggcgt cgtccggcgt gcggatgaag tgcttgatcg
     1681 tcgggagcgg catgaactcc gtgagccagt tgaggcggac catctcgtcc gtgacgtcgt
     1741 tggcgaccga gcccttgccg tgcttgagga agagctccgg ggcgtccggc ttgccgtaga
     1801 ggcggtagat cgtggcgccc gactggccga cgttgtcgcg ggaccacttg tagccgtaga
     1861 ggtcggcgtc catgttcgag ttgaggcgcg ggcgcgagac gtgcgtcttc tccttgccca
     1921 tgaattctgc tgtgtaaggt gtgcgtgaga gagagggtcg tgagtgagtc ttgaagagaa
     1981 acgtcaaagc ttgaagcgta gcagaatacg gaaaacgact tgggggaatg gcgaggagcg
     2041 tcgcctttta tgctccaagc ggagacgatg aggccgatat tacagtggct tagtagcgtg
     2101 tacaatattt ccataaaagg ctacatggtg agtttttcac gtgaccgtaa ttagtcccaa
     2161 ttaggcttta ttgtatattg ctactaagca tcgttaaatg acctgctcga agcttcgtct
     2221 ataaaaggcg ggcgtgtgtg cgcttccccc gctacgtctt tttcacaaac actttgcgac
     2281 ttgaccaaga cctttcaccc gcaccaccct gttatcccta gcggatctgc cggtagaggt
     2341 gtggtcaata agagcgacct catactatac ctgagaaagc aacctgacct acaggaaaga
     2401 gttactcaag aataagaatt ttcgttttaa aacctaagag tcactttaaa atttgtatac
     2461 acttattttt tttataactt atttaataat aaaaatcata aatcataaga aattcgctta
     2521 tttagaagtg gcgctcagag gtgggcctcg agggtctcag attggcagat gtggtcgcgc
     2581 tcctcggtgt cgtccttaga ttgggtgtat tgcttgcgga tccagtggta agggacgtca
     2641 ggggcaggtt ggttgtggag aggcttgcag atggtgtttt ggtgggcctc gacgcactta
     2701 gacttgtcag agaggagagg gtagaggagg gtgacagggc actcgacgcc gtcgtcgcga
     2761 gggatgtgtt ggacgtcgtt agggagagag acgttcatgt cgccggtgag gacaggagag
     2821 ttctccttga agtcttggcc ggtgaactcg acgatgttgt ggtaggtgcc gttcttcatg
     2881 aagtgttggt gcttggcgtg gatagagccg tcgcccttgt attggatgtg gcggtcgtag
     2941 gtgaagccct caggcatgac ctcgtggaac cagttcttga ggccagaagg gtacttggtg
     3001 tagtacttca tgccgtagcc gaaagaggtg ccgagggcgg cccaagacat agggagcttg
     3061 ccagaggtgc agacgtactt gcccttgtga gagccctcgt gagagttgcc ctcgccctcg
     3121 ccctcgacgg tgaaagactt gccgttgatg gtgcccttga gttggaactt gaaaggggta
     3181 gaggcagaac cagaaccagc accgtcaccg atccgtcgac ctgcagcgta cgaagcttca
     3241 gctggcggcc gcgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg
     3301 tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc
     3361 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     3421 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3481 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     3541 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3601 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3661 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3721 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3781 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3841 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3901 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3961 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4021 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4081 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4141 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4201 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4261 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4321 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4381 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4441 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4501 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4561 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4621 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4681 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4741 atcaaaggat cttc
//
