LOCUS       sequence-468369-        4577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            mNeonGreen and selection on Nat..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4577)
  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 4577)
  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-mNeonGreen-Nat
FEATURES             Location/Qualifiers
     source          1..4577
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      466..653
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(521..545)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             1062..1634
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1640..1837
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(1880..1899)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1881..1899)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1986..2003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2157..2745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2237..2256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2916..3707,3708..3776))
                     /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     3539..3558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3777..3881)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3949..3967
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4005..4027)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4127..4146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4227..4245
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4227..4244
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      join(4570..4577,1..2)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ggtcgacggc tctggctacc aagtccaccg caccatgcaa ttcgaggacg gcgcctctct
       61 cactgtcaac tacagataca cctacgaggg ctctcacatc aagggcgagg cccaagtcaa
      121 gggcaccggc ttccctgccg acggccctgt catgaccaac tctctcaccg ccgccgactg
      181 gtgccgctct aagaagacct accctaacga caagaccatc atctctacct tcaagtggtc
      241 ttacaccacc ggcaacggca agcgctaccg ctctaccgcc cgcaccacct acaccttcgc
      301 caagcctatg gccgccaact acctcaagaa ccaacctatg tacgtcttcc gcaagaccga
      361 gctcaagcac tctaagaccg agctcaactt caaggagtgg caaaaggcct tcaccgacgt
      421 catgggcatg gacgagctct acaagtgagc gccacttcta aataagcgaa tttcttatga
      481 tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt
      541 gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg
      601 ttgctttctc aggtatagta tgaggtcgct cttattgacc acacctctac cggcagatcc
      661 gctagggata acagggtggt gcgggtgaaa ggtcttggtc aagtcgcaaa gtgtttgtga
      721 aaaagacgta gcgggggaag cgcacacacg cccgcctttt atagacgaag cttcgagcag
      781 gtcatttaac gatgcttagt agcaatatac aataaagcct aattgggact aattacggtc
      841 acgtgaaaaa ctcaccatgt agccttttat ggaaatattg tacacgctac taagccactg
      901 taatatcggc ctcatcgtct ccgcttggag cataaaaggc gacgctcctc gccattcccc
      961 caagtcgttt tccgtattct gctacgcttc aagctttgac gtttctcttc aagactcact
     1021 cacgaccctc tctctcacgc acaccttaca cagcagaatt catgggtacc actcttgacg
     1081 acacggctta ccggtaccgc accagtgtcc cgggggacgc cgaggccatc gaggcactgg
     1141 atgggtcctt caccaccgac accgtcttcc gcgtcaccgc caccggggac ggcttcaccc
     1201 tgcgggaggt gccggtggac ccgcccctga ccaaggtgtt ccccgacgac gaatcggacg
     1261 acgaatcgga cgacggggag gacggcgacc cggactcccg gacgttcgtc gcgtacgggg
     1321 acgacggcga cctggcgggc ttcgtggtcg tctcgtactc cggctggaac cgccggctga
     1381 ccgtcgagga catcgaggtc gccccggagc accgggggca cggggtcggg cgcgcgttga
     1441 tggggctcgc gacggagttc gcccgcgagc ggggcgccgg gcacctctgg ctggaggtca
     1501 ccaacgtcaa cgcaccggcg atccacgcgt accggcggat ggggttcacc ctctgcggcc
     1561 tggacaccgc cctgtacgac ggcaccgcct cggacggcga gcaggcgctc tacatgagca
     1621 tgccctgccc ctaatcagta ctgacaataa aaagattctt gttttcaaga acttgtcatt
     1681 tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg tgatttatat
     1741 tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc
     1801 atgcgtcaat cgtatgtgaa tgctggtcgc tatactgcag atccactagt ggcctatgcg
     1861 gccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac
     1921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1981 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2041 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2101 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2161 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2221 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2281 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2341 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2401 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2461 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2521 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2581 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2641 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2701 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2761 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2821 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2881 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2941 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3001 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3061 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3121 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3181 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3241 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3301 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3361 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3421 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3481 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3541 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3601 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3661 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3721 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3781 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3841 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3901 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     3961 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     4021 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4081 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     4141 attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac
     4201 ataaccttat gtatcataca catacgattt aggtgacact atagaacgcg gccgccagct
     4261 gaagcttcgt acgctgcagg tcgacggatc ggtgacggtg ctggttctgg ttctatggtc
     4321 tctaagggcg aggaggacaa catggcctct ctccctgcca cccacgagct ccacatcttc
     4381 ggctctatca acggcgtcga cttcgacatg gtcggccaag gcaccggcaa ccctaacgac
     4441 ggctacgagg agctcaacct caagtctacc aagggcgacc tccaattctc tccttggatc
     4501 ctcgtccctc acatcggcta cggcttccac caatacctcc cttaccctga cggcatgtct
     4561 cctttccaag ccgccat
//
