LOCUS       sequence-468392-        4823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for deleting or C-terminally tagging endogenous genes with 
            sfGFP and selection on G418..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4823)
  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 4823)
  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-G418
FEATURES             Location/Qualifiers
     source          1..4823
                     /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(join(2535..3248,3249..3251,3252..3254))
                     /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(3324..3342)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3325..3342)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3423..3442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3542..3564
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3602..3620)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3688..3792
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3793..3861,3862..4653)
                     /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(4011..4030)
                     /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 gcgctcactt gtagagctcg tccatgccgt gggtgatgcc ggcggcggtg
     2581 acgaactcga ggaggaccat gtggtcgcgc ttctcgttag ggtccttaga gaggacagat
     2641 tgggtagaga ggtagtggtt gtcagggagg aggacagggc cgtcgccgat aggggtgttt
     2701 tgttggtagt ggtcggcgag ttggacagag ccgtcctcga cgttgtggcg gatcttgaag
     2761 ttggccttga tgccgttctt ttgcttgtcg gcggtgatgt agacgttgtg agagttgaag
     2821 ttgtactcga gcttgtggcc gaggatgttg ccgtcctcct tgaagtcgat gcccttgagc
     2881 tcgatgcggt tgacgagggt gtcgccctcg aacttgacct cggcgcgggt cttgtaggtg
     2941 ccgtcgtcct tgaaagagat ggtgcgctct tggacgtagc cctcaggcat ggcagacttg
     3001 aagaagtcgt ggcgcttcat gtggtcaggg tagcgagaga agcattggac gccgtaggtg
     3061 agggtggtga cgagggtagg ccaagggaca gggagcttgc cggtggtgca gatgaacttg
     3121 agggtgagct tgccgttggt ggcgtcgccc tcgccctcgc cgcggacaga gaacttgtgg
     3181 ccgttgacgt cgccgtcgag ctcgacgagg atagggacga cgccggtgaa gagctcctcg
     3241 cccttagaga ccatagaacc agaaccagca ccgtcaccga tccgtcgacc tgcagcgtac
     3301 gaagcttcag ctggcggccg cgttctatag tgtcacctaa atcgtatgtg tatgatacat
     3361 aaggttatgt attaattgta gccgcgttct aacgacaata tgtccatatg gtgcactctc
     3421 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     3481 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3541 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     3601 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     3661 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3721 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3781 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3841 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3901 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3961 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4021 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4081 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4141 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4201 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4261 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4321 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4381 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4441 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4501 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4561 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4621 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4681 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4741 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4801 gtagaaaaga tcaaaggatc ttc
//
