LOCUS       sequence-350051-        7567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeted gene knock-in plasmid for Schizophyllum commune.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7567)
  AUTHORS   Vonk PJ, Ohm RA
  TITLE     Targeted gene knock-in reduces variation between transformants in 
            the mushroom-forming fungus Schizophyllum commune
  JOURNAL   Open Res Europe 2021, 1:140
REFERENCE   2  (bases 1 to 7567)
  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: "Open Res 
            Europe 2021, 1:140"
COMMENT     Sequence Label: pPV033
FEATURES             Location/Qualifiers
     source          1..7567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(471..488)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(471..487)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(480..502)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(696..715)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     815..837
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(875..893)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        961..1065
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1066..1134,1135..1926)
                     /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(1284..1303)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2097..2685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2586..2605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2839..2856
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2973..2994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3009..3014,3015..3032,3033..3039)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3047..3063
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3052..3074
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3071..3087
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3071..3087
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(4669..5238)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDVEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     CDS             7181..7555
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
ORIGIN
        1 aacaccgccg gtccgacgcg gcccgacggg tccgaggggg taccgcggcc gccttaagac
       61 gcgtggatcg atccccggca tcgcgatcag caagagcttc gtgaagctca ttgcctggta
      121 cgacaacgag tggggctact cccgccgtgt ctgcgacctg ctcgtctacg cggcgaagca
      181 ggacggcgcg ctctaagcgg cgaagcaaga cggcgcgctc taagtgcctg atcctttgga
      241 cgcttgtact ttacctctac atagactggg ccggagagta aaagaagaaa ggaatgtaac
      301 gttgtacaac atgtctaata attgtacgat cccaaaaatg atagaatgga tggtttgaaa
      361 gtcgtgcatg tatgcgatag gggccacctc gaaggaattt cgcgggcgta cggccagaac
      421 ctctttggag caagctggga attcggcgcg tgggtaccga gctcgaattc actggccgtc
      481 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
      541 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
      601 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
      661 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
      721 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
      781 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
      841 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
      901 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
      961 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1021 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1081 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1141 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1201 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1261 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     1321 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1381 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1441 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     1501 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     1561 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     1621 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     1681 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     1741 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     1801 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     1861 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     1921 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     1981 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2041 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2101 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2161 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2221 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     2281 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2341 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2401 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2461 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2521 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2581 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2641 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2701 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2761 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2821 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     2881 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     2941 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     3001 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     3061 caatttcaca caggaaacag ctatgaccat gattacgcca gccaacaatc agatccgcta
     3121 gcgtctaacg tcgtaaaaca acctcccact tgacctgagc ctggggtcaa gtcactccgt
     3181 gaagccccgc gctacgtgaa gctagacccc accttgcgcg aagcgagggc aagtcccggg
     3241 tattgttctg tggcacgtag gcttcgtcgg gattgatgcc gccgtgctga ggggatgcgc
     3301 cacaatagca aagaacccgg atgtttggcg actttgcgtg cggtgagggc tcaggtcgat
     3361 gttggcaggg agctgttgtc gttatccttc gactagattg cgagagagta ggcgcagaga
     3421 atcagtgaga accgatgggt aaatcgtacg gtcaaaaatg gcctattcga caatattgga
     3481 gcctattcaa gcacctcgct gtacctcgta gagccatcct cgcagatcca gctccctaac
     3541 gataatgtgg aggaaacacg ggctactgct acctctcccc cttcccctga taggagtaag
     3601 aggacatcaa gagcagtatg gagacatcga gaggggtaag gggacatcat gaggggtatg
     3661 gaaccatcac ggagagccag atcgttcagt tcagttgcag acgatcccag ttcagtcgta
     3721 tacgatccca gctcagttgc agatgatcga ctcgagtcaa gcgagtttga gggactgtat
     3781 gccatcgggc gagggggtcc aggagatata cgcgtatggc agcgtcgccg cgaaatgaac
     3841 atccgaacgt catgcacgtc ggagccgagc gagtggcgta gatagtcctg agcccaagtc
     3901 cgaagttcac gcaagctgac aaggcgtgag gtcaagaggt cgatgcacgc ggagaggtcg
     3961 atatctgcag gccgtccagt ctaaggggcc aggttcagag accatcgtgg acattgctcc
     4021 ttggggcagc gagcgtaagc gacagccaga aaggggtctc gtacgtcctt ctcgtcgctg
     4081 agaacaaagc aagcggtcgc agaatccaga aaaaacatgt gaggggttcg cgcgtgtgag
     4141 tcataagcac cgaggttgct ccatggcccg gcatggtggt ctgggaggtg gaaggatggt
     4201 aagaaaacgt ccacctttca ccctcctttt gtaccccctt acttcgggac ctgaggcttc
     4261 attcatgacg tagactggca tcctcgccgt aaagtggagt gctttagtcc acgactcagc
     4321 ctttacttag gttcgccatg tcgtcatgcg gttgttgcgt ggcgaagttc aaccatgcac
     4381 gtgtggggtc gcgtgactga gcggatagag aacaaacaac aacaacaatg ttcattatcg
     4441 agggaatgag tcaacacctt ggagtatgta gcaagacgat cacttgtccg cgaccctatg
     4501 acatcgaagg gattccgaat ttgctaggca gtaacgaggc tcagattgcc cgaggctccc
     4561 gtcgcccgaa tatcccaccg ataggcgcgt tcacctgcgg atctcttgag tagtaggtca
     4621 gagcgtgtct cgcattgtct agactgacgt gcactcacag gatcgatcct aggggcaggg
     4681 catgctcatg tagagcgcct gctcgccgtc cgaggcggtg ccgtcgtaca gggcggtgtc
     4741 caggccgcag agggtgaacc ccatccgccg gtacgcgtgg atcgccggtg cgttgacgtt
     4801 ggtgacctcc agccagaggt gcccggcgcc ccgctcgcgg gcgaactccg tcgcgagccc
     4861 catcaacgcg cgcccgaccc cgtgcccccg gtgctccggg gcgacctcga cgtcctcgac
     4921 ggtcagccgg cggttccagc cggagtacga gacgaccacg aagcccgcca ggtcgccgtc
     4981 gtccccgtac gcgacgaacg tccgggagtc cgggtcgccg tcctccccgt cgtccgattc
     5041 gtcgtccgat tcgtcgtcgg ggaacacctt ggtcaggggc gggtccaccg gcacctcccg
     5101 cagggtgaag ccgtccccgg tggcggtgac gcggaagacg gtgtcggtgg tgaaggaccc
     5161 atccagtgcc tcgatggcct cggcgtcccc cgggacactg gtgcggtacc ggtaagccgt
     5221 gtcgtcaaga gtggtcatgg tgatgggcat tatgtgtgat gggatgcgat gggagaggga
     5281 agtgctctgg atgggagtgc tggagaaaga gggagacggc gggcggcgcg cgcgcctttt
     5341 atacccacgc ccgaaagatc cgatcgatac tgacaaaacg ggatgaacac atcggcggcg
     5401 gcctggactg cgcgccatct gcaaatgccc agccagtccc gtcgggcgcc accaccagcc
     5461 ctggtcgagt ccccctcgag gcgcgccggg gtcctttctg acgatgacca ccgactcggc
     5521 cccttgcgat gagcaagcag caccgagccg gcgatatcaa cgcgaagagg tcggtgatgt
     5581 cctcgcgcag ccgtcgtaat ggacgtgctg atggcggcga agacgacgga ggagcaccgt
     5641 tgggtccttc ccggttgggt tgccgatgct caggttgcgg gacgtcttcc gtcgattgac
     5701 agaggtggat gacgctgcgg ttagccacgc gagggcaacg tgaagagggt ggcatgtttg
     5761 agcaagcgtg agtcaccgcg gtagccaata gtctgagagg cccaagctga aaggcgggtc
     5821 cagtcttgag gcatcgtgag cgaggggtcc ttttagacaa tgccgtagtt ggagagctcg
     5881 cgcgcgtgac ggcagtcatg ttgagcgagg tcatagcaca aatcagaggt ttgaagcgcc
     5941 tcggcccccc agagacagag ggacgacaca gagggaaagc aatcagcaag tatgaagtac
     6001 cttcgcagat taaggactga gtcccctggt gggattccat cgtaggatcc gtcaagagag
     6061 taacccgcag aagaagattt tggtagacca ttgctggggc gagagatgcg agggagggaa
     6121 gaaaagagtt gcgacgagcg ccagagcaag ttcttataaa gtgggcttgt acccacctcg
     6181 gtccagcgcc gcggaggact gcccatacag tggcgccgag tcagcgctca tgttcccatt
     6241 tctgtgtgat acagatagca cctcggtaag ctcctgagct ctgaggaggt agaagagagc
     6301 tcgcggcgag cgcccgcggg cagcgcccat agcgggagag gtcagatgca cgccggcacg
     6361 cctgtgccgc gcaataaccg ggaaaaggag cttgaattct gtcgccgcat tgtaccgcgt
     6421 aggtgcggag ataactcagc agaatattat ctgttcaagc ttgagatgtc cggaaggaca
     6481 tgcgctggtc tgcagaagag gtcaagagac gcacagtggt ggcagaaggc aaggtacaga
     6541 cggcgaaaag tcacgtcgtt ctgctgaagg ttatctgact tcccacacag gcgcacgctc
     6601 accttattac gcttagcaac gcacacgcgc tcatcggact tctacacaca agaaatccca
     6661 caattgcata ggattggact ggttacggac ttcactggac ttgcatggac ttcacgattt
     6721 gttgtacccg ctttgttgtt gtcgagggtg tcttgttgct gttgtatgag taatgttgaa
     6781 cacattcatt ggttgactgg cttccaatgg tctgtctgag gagtaagcaa tactgcgaag
     6841 agcagaagga gtgagagacc tacgtggtaa agcgcacaac agagctttgc acaggcacag
     6901 ccgtcagcct ttcaaacctg acctcgtatt taaatctcga gggggactcg accagggctg
     6961 gtggtggcgc ccgacgggac tggctgggca tttgcagatg gcgcgcagtc caggccgccg
     7021 ccgatgtgtt catcccgttt tgtcagtatc gatcggatct ttcgggcgtg ggtataaaag
     7081 gcgcgcgcgc cgcccgccgt ctccctcttt ctccagcact cccatccaga gcacttccct
     7141 ctcccatcgc atcccatcac acataatgcc catcaccaac atggccaagt tgaccagtgc
     7201 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
     7261 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
     7321 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
     7381 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
     7441 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
     7501 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgaccgac
     7561 gccgacc
//
