LOCUS       sequence-468399-        4578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            mScarlet and selection on Nat..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4578)
  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 4578)
  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-mScarlet-Nat
FEATURES             Location/Qualifiers
     source          1..4578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      546..1134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1035..1054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1288..1305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1392..1411
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1392..1410
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1454..1651)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1657..2229)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      complement(2638..2825)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2746..2770
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(3624..3642)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3625..3642)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3723..3742)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3842..3864
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3902..3920)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3988..4092
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4093..4161,4162..4578,1..375)
                     /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(4311..4330)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
       61 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
      121 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
      181 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
      241 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
      301 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
      361 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
      421 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
      481 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
      541 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
      601 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
      661 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
      721 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
      781 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      841 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      901 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      961 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1021 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1081 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     1141 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     1201 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     1261 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     1321 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag gttaacctgg
     1381 cttatcgaaa ttaatacgac tcactatagg gagaccggca gatccgcggc cgcataggcc
     1441 actagtggat ctgcagtata gcgaccagca ttcacatacg attgacgcat gatattactt
     1501 tctgcgcact taacttcgca tctgggcaga tgatgtcgag gcgaaaaaaa atataaatca
     1561 cgctaacatt tgattaaaat agaacaacta caatataaaa aaactataca aatgacaagt
     1621 tcttgaaaac aagaatcttt ttattgtcag tactgattag gggcagggca tgctcatgta
     1681 gagcgcctgc tcgccgtccg aggcggtgcc gtcgtacagg gcggtgtcca ggccgcagag
     1741 ggtgaacccc atccgccggt acgcgtggat cgccggtgcg ttgacgttgg tgacctccag
     1801 ccagaggtgc ccggcgcccc gctcgcgggc gaactccgtc gcgagcccca tcaacgcgcg
     1861 cccgaccccg tgcccccggt gctccggggc gacctcgatg tcctcgacgg tcagccggcg
     1921 gttccagccg gagtacgaga cgaccacgaa gcccgccagg tcgccgtcgt ccccgtacgc
     1981 gacgaacgtc cgggagtccg ggtcgccgtc ctccccgtcg tccgattcgt cgtccgattc
     2041 gtcgtcgggg aacaccttgg tcaggggcgg gtccaccggc acctcccgca gggtgaagcc
     2101 gtccccggtg gcggtgacgc ggaagacggt gtcggtggtg aaggacccat ccagtgcctc
     2161 gatggcctcg gcgtcccccg ggacactggt gcggtaccgg taagccgtgt cgtcaagagt
     2221 ggtacccatg aattctgctg tgtaaggtgt gcgtgagaga gagggtcgtg agtgagtctt
     2281 gaagagaaac gtcaaagctt gaagcgtagc agaatacgga aaacgacttg ggggaatggc
     2341 gaggagcgtc gccttttatg ctccaagcgg agacgatgag gccgatatta cagtggctta
     2401 gtagcgtgta caatatttcc ataaaaggct acatggtgag tttttcacgt gaccgtaatt
     2461 agtcccaatt aggctttatt gtatattgct actaagcatc gttaaatgac ctgctcgaag
     2521 cttcgtctat aaaaggcggg cgtgtgtgcg cttcccccgc tacgtctttt tcacaaacac
     2581 tttgcgactt gaccaagacc tttcacccgc accaccctgt tatccctagc ggatctgccg
     2641 gtagaggtgt ggtcaataag agcgacctca tactatacct gagaaagcaa cctgacctac
     2701 aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc actttaaaat
     2761 ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa tcataagaaa
     2821 ttcgcttatt tagaagtggc gctctagatt cacttgtaga gctcgtccat gccgccggta
     2881 gagtggcggc cctcagagcg ctcgtattgc tcgacgacgg tgtagtcctc gttgtgagag
     2941 gtgatgtcga gcttgcggtc gacgttgtag gcgccaggca tttggacagg cttcttggcc
     3001 ttgtaggtgg tcttgaagtc ggcgaggtag cggccgccgt ccttgaggcg gagggccatc
     3061 ttgatgtcgc ccttgaggac gccgtcctca gggtagaggc gctcggtaga ggcctcccag
     3121 cccattgtct tcttttgcat gacagggccg tcaggaggga agttggtgcc gcggagcttg
     3181 accttgtaga tgagggtgcc gtcctcgaga gaggtgtctt gggtgacggt gacggcgccg
     3241 ccgtcctcga agttcatgac gcgctcccac ttgaagccct cagggaaaga ttgcttgtag
     3301 tagtcaggga tgtcggcagg gtgcttggtg aaggcgcgag agccgtacat gaattgagga
     3361 gagaggatgt cccaagagaa agggagaggg ccgcccttgg tgaccttgag cttggcggtt
     3421 tgggtgccct cgtaagggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc
     3481 atagagccct ccatgtggac cttgaagcgc atgaactcct tgatgacggc ctcgccctta
     3541 gagaccatgg taccagaacc agaaccagca ccgtcaccga tccgtcgacc tgcagcgtac
     3601 gaagcttcag ctggcggccg cgttctatag tgtcacctaa atcgtatgtg tatgatacat
     3661 aaggttatgt attaattgta gccgcgttct aacgacaata tgtccatatg gtgcactctc
     3721 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     3781 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3841 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     3901 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     3961 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4021 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4081 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4141 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4201 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4261 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4321 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4381 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4441 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4501 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4561 gtaactcgcc ttgatcgt
//
