LOCUS       sequence-468382-        5033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            mNeonGreen and selection on Hyg..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5033)
  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 5033)
  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-Hyg
FEATURES             Location/Qualifiers
     source          1..5033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      121..130
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     terminator      594..781
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(649..673)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             1190..2218
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1190..2218
                     /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      2224..2421
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2464..2483)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2465..2483)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2570..2587)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2741..3329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2821..2840)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3500..4291,4292..4360))
                     /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     4123..4142
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4361..4465)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4533..4551
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4589..4611)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4711..4730
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4811..4829
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4811..4828
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gagctcaacc tcaagtctac caagggcgac ctccaattct ctccttggat cctcgtccct
       61 cacatcggct acggcttcca ccaatacctc ccttaccctg acggcatgtc tcctttccaa
      121 gccgccatgg tcgacggctc tggctaccaa gtccaccgca ccatgcaatt cgaggacggc
      181 gcctctctca ctgtcaacta cagatacacc tacgagggct ctcacatcaa gggcgaggcc
      241 caagtcaagg gcaccggctt ccctgccgac ggccctgtca tgaccaactc tctcaccgcc
      301 gccgactggt gccgctctaa gaagacctac cctaacgaca agaccatcat ctctaccttc
      361 aagtggtctt acaccaccgg caacggcaag cgctaccgct ctaccgcccg caccacctac
      421 accttcgcca agcctatggc cgccaactac ctcaagaacc aacctatgta cgtcttccgc
      481 aagaccgagc tcaagcactc taagaccgag ctcaacttca aggagtggca aaaggccttc
      541 accgacgtca tgggcatgga cgagctctac aagtgagcgc cacttctaaa taagcgaatt
      601 tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat
      661 tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt
      721 aggtcaggtt gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg
      781 gcagatccgc tagggataac agggtggtgc gggtgaaagg tcttggtcaa gtcgcaaagt
      841 gtttgtgaaa aagacgtagc gggggaagcg cacacacgcc cgccttttat agacgaagct
      901 tcgagcaggt catttaacga tgcttagtag caatatacaa taaagcctaa ttgggactaa
      961 ttacggtcac gtgaaaaact caccatgtag ccttttatgg aaatattgta cacgctacta
     1021 agccactgta atatcggcct catcgtctcc gcttggagca taaaaggcga cgctcctcgc
     1081 cattccccca agtcgttttc cgtattctgc tacgcttcaa gctttgacgt ttctcttcaa
     1141 gactcactca cgaccctctc tctcacgcac accttacaca gcagaattca tgggtaaaaa
     1201 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
     1261 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
     1321 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
     1381 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt
     1441 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
     1501 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc
     1561 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     1621 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     1681 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     1741 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     1801 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     1861 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     1921 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg
     1981 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     2041 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     2101 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga
     2161 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaataatc
     2221 agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata gtttttttat
     2281 attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt tcgcctcgac
     2341 atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg
     2401 tgaatgctgg tcgctatact gcagatccac tagtggccta tgcggccgcg gatctgccgg
     2461 tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat taatgaatcg
     2521 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3181 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3421 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3481 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3541 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3601 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3661 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3721 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3781 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3841 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3901 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3961 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4021 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4081 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4141 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4201 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4261 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4321 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4381 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4441 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     4501 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     4561 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     4621 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     4681 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     4741 accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca
     4801 tacacatacg atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgctg
     4861 caggtcgacg gatcggtgac ggtgctggtt ctggttctat ggtctctaag ggcgaggagg
     4921 acaacatggc ctctctccct gccacccacg agctccacat cttcggctct atcaacggcg
     4981 tcgacttcga catggtcggc caaggcaccg gcaaccctaa cgacggctac gag
//
