LOCUS       sequence-468368-        4586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            sfGFP and selection on Nat..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4586)
  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 4586)
  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-sfGFP-Nat
FEATURES             Location/Qualifiers
     source          1..4586
                     /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"
     CDS             complement(1112..1684)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      complement(2093..2280)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2201..2225
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(join(2298..3011,3012..3014,3015..3017))
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     promoter        complement(3087..3105)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3088..3105)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3186..3205)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3305..3327
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3365..3383)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3451..3555
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3556..3624,3625..4416)
                     /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(3774..3793)
                     /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 attaggggca gggcatgctc atgtagagcg
     1141 cctgctcgcc gtccgaggcg gtgccgtcgt acagggcggt gtccaggccg cagagggtga
     1201 accccatccg ccggtacgcg tggatcgccg gtgcgttgac gttggtgacc tccagccaga
     1261 ggtgcccggc gccccgctcg cgggcgaact ccgtcgcgag ccccatcaac gcgcgcccga
     1321 ccccgtgccc ccggtgctcc ggggcgacct cgatgtcctc gacggtcagc cggcggttcc
     1381 agccggagta cgagacgacc acgaagcccg ccaggtcgcc gtcgtccccg tacgcgacga
     1441 acgtccggga gtccgggtcg ccgtcctccc cgtcgtccga ttcgtcgtcc gattcgtcgt
     1501 cggggaacac cttggtcagg ggcgggtcca ccggcacctc ccgcagggtg aagccgtccc
     1561 cggtggcggt gacgcggaag acggtgtcgg tggtgaagga cccatccagt gcctcgatgg
     1621 cctcggcgtc ccccgggaca ctggtgcggt accggtaagc cgtgtcgtca agagtggtac
     1681 ccatgaattc tgctgtgtaa ggtgtgcgtg agagagaggg tcgtgagtga gtcttgaaga
     1741 gaaacgtcaa agcttgaagc gtagcagaat acggaaaacg acttggggga atggcgagga
     1801 gcgtcgcctt ttatgctcca agcggagacg atgaggccga tattacagtg gcttagtagc
     1861 gtgtacaata tttccataaa aggctacatg gtgagttttt cacgtgaccg taattagtcc
     1921 caattaggct ttattgtata ttgctactaa gcatcgttaa atgacctgct cgaagcttcg
     1981 tctataaaag gcgggcgtgt gtgcgcttcc cccgctacgt ctttttcaca aacactttgc
     2041 gacttgacca agacctttca cccgcaccac cctgttatcc ctagcggatc tgccggtaga
     2101 ggtgtggtca ataagagcga cctcatacta tacctgagaa agcaacctga cctacaggaa
     2161 agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta
     2221 tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc
     2281 ttatttagaa gtggcgctca cttgtagagc tcgtccatgc cgtgggtgat gccggcggcg
     2341 gtgacgaact cgaggaggac catgtggtcg cgcttctcgt tagggtcctt agagaggaca
     2401 gattgggtag agaggtagtg gttgtcaggg aggaggacag ggccgtcgcc gataggggtg
     2461 ttttgttggt agtggtcggc gagttggaca gagccgtcct cgacgttgtg gcggatcttg
     2521 aagttggcct tgatgccgtt cttttgcttg tcggcggtga tgtagacgtt gtgagagttg
     2581 aagttgtact cgagcttgtg gccgaggatg ttgccgtcct ccttgaagtc gatgcccttg
     2641 agctcgatgc ggttgacgag ggtgtcgccc tcgaacttga cctcggcgcg ggtcttgtag
     2701 gtgccgtcgt ccttgaaaga gatggtgcgc tcttggacgt agccctcagg catggcagac
     2761 ttgaagaagt cgtggcgctt catgtggtca gggtagcgag agaagcattg gacgccgtag
     2821 gtgagggtgg tgacgagggt aggccaaggg acagggagct tgccggtggt gcagatgaac
     2881 ttgagggtga gcttgccgtt ggtggcgtcg ccctcgccct cgccgcggac agagaacttg
     2941 tggccgttga cgtcgccgtc gagctcgacg aggataggga cgacgccggt gaagagctcc
     3001 tcgcccttag agaccataga accagaacca gcaccgtcac cgatccgtcg acctgcagcg
     3061 tacgaagctt cagctggcgg ccgcgttcta tagtgtcacc taaatcgtat gtgtatgata
     3121 cataaggtta tgtattaatt gtagccgcgt tctaacgaca atatgtccat atggtgcact
     3181 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     3241 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3301 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     3361 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     3421 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3481 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3541 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3601 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3661 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3721 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3781 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3841 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3901 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3961 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4021 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4081 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4141 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4201 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4261 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4321 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4381 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4441 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4501 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4561 cccgtagaaa agatcaaagg atcttc
//
