LOCUS       sequence-428736-        8362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fuses insert of interest to an HA-tag, stop codon and S. commune 
            Hom2 terminator, with flanks directing the construct to S. commune 
            targeted integration locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8362)
  TITLE     Targeted gene knock-in plasmid for Schizophyllum commune, including 
            HA-tag
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8362)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pEB018
FEATURES             Location/Qualifiers
     source          1..8362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..37)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(20..36)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(29..51)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(245..264)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(424..442)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        510..614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(615..683,684..1475)
                     /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(833..852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1646..2234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2135..2154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2388..2405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2522..2543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2558..2563,2564..2581,2582..2588)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2596..2612
                     /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     2601..2623
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2620..2636
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2620..2636
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3716..3796
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(5013..5582)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDVEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     CDS             7525..7899
                     /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 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
       61 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
      121 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
      181 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
      241 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
      301 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
      361 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
      421 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
      481 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      541 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      601 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
      661 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
      721 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
      781 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
      841 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
      901 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
      961 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1021 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1081 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1141 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1201 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1261 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1321 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1381 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1441 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1501 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1561 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1621 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     1801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2281 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2341 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2401 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2461 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     2521 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     2581 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     2641 attacgccag ccaacaatca gatccgctag cgtctaacgt cgtaaaacaa cctcccactt
     2701 gacctgagcc tggggtcaag tcactccgtg aagccccgcg ctacgtgaag ctagacccca
     2761 ccttgcgcga agcgagggca agtcccgggt attgttctgt ggcacgtagg cttcgtcggg
     2821 attgatgccg ccgtgctgag gggatgcgcc acaatagcaa agaacccgga tgtttggcga
     2881 ctttgcgtgc ggtgagggct caggtcgatg ttggcaggga gctgttgtcg ttatccttcg
     2941 actagattgc gagagagtag gcgcagagaa tcagtgagaa ccgatgggta aatcgtacgg
     3001 tcaaaaatgg cctattcgac aatattggag cctattcaag cacctcgctg tacctcgtag
     3061 agccatcctc gcagatccag ctccctaacg ataatgtgga ggaaacacgg gctactgcta
     3121 cctctccccc ttcccctgat aggagtaaga ggacatcaag agcagtatgg agacatcgag
     3181 aggggtaagg ggacatcatg aggggtatgg aaccatcacg gagagccaga tcgttcagtt
     3241 cagttgcaga cgatcccagt tcagtcgtat acgatcccag ctcagttgca gatgatcgac
     3301 tcgagtcaag cgagtttgag ggactgtatg ccatcgggcg agggggtcca ggagatatac
     3361 gcgtatggca gcgtcgccgc gaaatgaaca tccgaacgtc atgcacgtcg gagccgagcg
     3421 agtggcgtag atagtcctga gcccaagtcc gaagttcacg caagctgaca aggcgtgagg
     3481 tcaagaggtc gatgcacgcg gagaggtcga tatctgcagg ccgtccagtc taaggggcca
     3541 ggttcagaga ccatcgtgga cattgctcct tggggcagcg agcgtaagcg acagccagaa
     3601 aggggtctcg tacgtccttc tcgtcgctga gaacaaagca agcggtcgca gaatccagaa
     3661 aaaacatgtg aggggttcgc gcgtgtgagt cataagcacc gaggttgctc actagtaccc
     3721 ctacgacgtc cctgattatg cgtaccctta tgatgtgccc gactacgcct atccctatga
     3781 cgtcccggac tatgcttgag gatcctgtga gtgcacgtca gtctagacaa tgcgagacac
     3841 gctctgacct actactcaac ccctggcaat catttgctgc tcgccaacca gccgtctccg
     3901 atcctcactc tcaatcaaat caaccaacct taagcacact cactcgcctc tcttgttttc
     3961 cccttgcata tttaagccca cttacgcctc gcccgttgta cagctctgta tactaatgct
     4021 ttccttctcc tcttcacctc ttagtctccc accttcgctt cgccttgcgc gtgcgcaccc
     4081 ttcacccttc caaccttcaa caccaacacg gacctgcggt ttctttattc cttctttcat
     4141 tcgccttctt cgttatttcc ttttggcctg cctttatctt attgtactca ttttttctca
     4201 ctgtctctct cgtgcactca acgcgctgct actggtgtct ccgccgtaca tatcttagag
     4261 cttgtccata atcgtccgtc tcgtcccgct agttttgcct gtacacttct gcaggttacc
     4321 tttcacccgt actcatcaac atctggattt cgaggaagat tgcgcagcta tcgaatccca
     4381 ttccatgttt tgcatcgcag cacacaggtt cgccatcgca ctcagactcc ggtctatctt
     4441 cttcggacaa ggtccgggtg gccacgatct acccaaacag tgaggaagcg gtaaggccgt
     4501 gatgggatgg cccggcatgg tggtctggga ggtggaagga tggtaagaaa acgtccacct
     4561 ttcaccctcc ttttgtaccc ccttacttcg ggacctgagg cttcattcat gacgtagact
     4621 ggcatcctcg ccgtaaagtg gagtgcttta gtccacgact cagcctttac ttaggttcgc
     4681 catgtcgtca tgcggttgtt gcgtggcgaa gttcaaccat gcacgtgtgg ggtcgcgtga
     4741 ctgagcggat agagaacaaa caacaacaac aatgttcatt atcgagggaa tgagtcaaca
     4801 ccttggagta tgtagcaaga cgatcacttg tccgcgaccc tatgacatcg aagggattcc
     4861 gaatttgcta ggcagtaacg aggctcagat tgcccgaggc tcccgtcgcc cgaatatccc
     4921 accgataggc gcgttcacct gcggatctct tgagtagtag gtcagagcgt gtctcgcatt
     4981 gtctagactg acgtgcactc acaggatcga tcctaggggc agggcatgct catgtagagc
     5041 gcctgctcgc cgtccgaggc ggtgccgtcg tacagggcgg tgtccaggcc gcagagggtg
     5101 aaccccatcc gccggtacgc gtggatcgcc ggtgcgttga cgttggtgac ctccagccag
     5161 aggtgcccgg cgccccgctc gcgggcgaac tccgtcgcga gccccatcaa cgcgcgcccg
     5221 accccgtgcc cccggtgctc cggggcgacc tcgacgtcct cgacggtcag ccggcggttc
     5281 cagccggagt acgagacgac cacgaagccc gccaggtcgc cgtcgtcccc gtacgcgacg
     5341 aacgtccggg agtccgggtc gccgtcctcc ccgtcgtccg attcgtcgtc cgattcgtcg
     5401 tcggggaaca ccttggtcag gggcgggtcc accggcacct cccgcagggt gaagccgtcc
     5461 ccggtggcgg tgacgcggaa gacggtgtcg gtggtgaagg acccatccag tgcctcgatg
     5521 gcctcggcgt cccccgggac actggtgcgg taccggtaag ccgtgtcgtc aagagtggtc
     5581 atggtgatgg gcattatgtg tgatgggatg cgatgggaga gggaagtgct ctggatggga
     5641 gtgctggaga aagagggaga cggcgggcgg cgcgcgcgcc ttttataccc acgcccgaaa
     5701 gatccgatcg atactgacaa aacgggatga acacatcggc ggcggcctgg actgcgcgcc
     5761 atctgcaaat gcccagccag tcccgtcggg cgccaccacc agccctggtc gagtccccct
     5821 cgaggcgcgc cggggtcctt tctgacgatg accaccgact cggccccttg cgatgagcaa
     5881 gcagcaccga gccggcgata tcaacgcgaa gaggtcggtg atgtcctcgc gcagccgtcg
     5941 taatggacgt gctgatggcg gcgaagacga cggaggagca ccgttgggtc cttcccggtt
     6001 gggttgccga tgctcaggtt gcgggacgtc ttccgtcgat tgacagaggt ggatgacgct
     6061 gcggttagcc acgcgagggc aacgtgaaga gggtggcatg tttgagcaag cgtgagtcac
     6121 cgcggtagcc aatagtctga gaggcccaag ctgaaaggcg ggtccagtct tgaggcatcg
     6181 tgagcgaggg gtccttttag acaatgccgt agttggagag ctcgcgcgcg tgacggcagt
     6241 catgttgagc gaggtcatag cacaaatcag aggtttgaag cgcctcggcc ccccagagac
     6301 agagggacga cacagaggga aagcaatcag caagtatgaa gtaccttcgc agattaagga
     6361 ctgagtcccc tggtgggatt ccatcgtagg atccgtcaag agagtaaccc gcagaagaag
     6421 attttggtag accattgctg gggcgagaga tgcgagggag ggaagaaaag agttgcgacg
     6481 agcgccagag caagttctta taaagtgggc ttgtacccac ctcggtccag cgccgcggag
     6541 gactgcccat acagtggcgc cgagtcagcg ctcatgttcc catttctgtg tgatacagat
     6601 agcacctcgg taagctcctg agctctgagg aggtagaaga gagctcgcgg cgagcgcccg
     6661 cgggcagcgc ccatagcggg agaggtcaga tgcacgccgg cacgcctgtg ccgcgcaata
     6721 accgggaaaa ggagcttgaa ttctgtcgcc gcattgtacc gcgtaggtgc ggagataact
     6781 cagcagaata ttatctgttc aagcttgaga tgtccggaag gacatgcgct ggtctgcaga
     6841 agaggtcaag agacgcacag tggtggcaga aggcaaggta cagacggcga aaagtcacgt
     6901 cgttctgctg aaggttatct gacttcccac acaggcgcac gctcacctta ttacgcttag
     6961 caacgcacac gcgctcatcg gacttctaca cacaagaaat cccacaattg cataggattg
     7021 gactggttac ggacttcact ggacttgcat ggacttcacg atttgttgta cccgctttgt
     7081 tgttgtcgag ggtgtcttgt tgctgttgta tgagtaatgt tgaacacatt cattggttga
     7141 ctggcttcca atggtctgtc tgaggagtaa gcaatactgc gaagagcaga aggagtgaga
     7201 gacctacgtg gtaaagcgca caacagagct ttgcacaggc acagccgtca gcctttcaaa
     7261 cctgacctcg tatttaaatc tcgaggggga ctcgaccagg gctggtggtg gcgcccgacg
     7321 ggactggctg ggcatttgca gatggcgcgc agtccaggcc gccgccgatg tgttcatccc
     7381 gttttgtcag tatcgatcgg atctttcggg cgtgggtata aaaggcgcgc gcgccgcccg
     7441 ccgtctccct ctttctccag cactcccatc cagagcactt ccctctccca tcgcatccca
     7501 tcacacataa tgcccatcac caacatggcc aagttgacca gtgccgttcc ggtgctcacc
     7561 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
     7621 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
     7681 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
     7741 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
     7801 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
     7861 ggcaactgcg tgcacttcgt ggccgaggag caggactgac cgacgccgac caacaccgcc
     7921 ggtccgacgc ggcccgacgg gtccgagggg gtaccgcggc cgccttaaga cgcgtggatc
     7981 gatccccggc atcgcgatca gcaagagctt cgtgaagctc attgcctggt acgacaacga
     8041 gtggggctac tcccgccgtg tctgcgacct gctcgtctac gcggcgaagc aggacggcgc
     8101 gctctaagcg gcgaagcaag acggcgcgct ctaagtgcct gatcctttgg acgcttgtac
     8161 tttacctcta catagactgg gccggagagt aaaagaagaa aggaatgtaa cgttgtacaa
     8221 catgtctaat aattgtacga tcccaaaaat gatagaatgg atggtttgaa agtcgtgcat
     8281 gtatgcgata ggggccacct cgaaggaatt tcgcgggcgt acggccagaa cctctttgga
     8341 gcaagctggg aattcggcgc gt
//
