LOCUS       sequence-189235-        6507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDONR split marker vector (GenBank accession number: KX904529) for 
            fungal gene deletion. Used with pSM1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6507)
  AUTHORS   Rahnama M, Forester N, Ariyawansa KG, Voisey CR, Johnson LJ, Johnson
            RD, Fleetwood DJ
  TITLE     Efficient targeted mutagenesis in Epichloe festucae using a split 
            marker system.
  JOURNAL   J Microbiol Methods. 2016 Dec 22. pii: S0167-7012(16)30368-2. doi: 
            10.1016/j.mimet.2016.12.017.
  PUBMED    28017692
REFERENCE   2  (bases 1 to 6507)
  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: "J Microbiol
            Methods. 2016 Dec 22. pii: S0167-7012(16)30368-2. doi: 
            10.1016/j.mimet.2016.12.017."
COMMENT     Sequence Label: pSM2
FEATURES             Location/Qualifiers
     source          1..6507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1470..2244
                     /gene="trpC"
                     /label=trpC terminator
                     /note="transcription terminator from the Aspergillus 
                     nidulans trpC gene (Punt et al., 1987)"
     primer_bind     2268..2290
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2282..2299
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2283..2299
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2316..2547
                     /gene="mutant version of attP"
                     /label=attP1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(2943..3248)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3045..3064)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3568..4251)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     4166..4185
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4252..4354)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(4499..4730)
                     /gene="mutant version of attP"
                     /label=attP2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4748..4767)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4749..4767)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4772..4788)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4772..4788)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4890..4909)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4901..5710
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4968..4987)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5857..6445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6346..6365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgcggcc gctcggaggg cgaagaatct cgtgctttca
      541 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
      601 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
      661 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
      721 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
      781 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
      841 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
      901 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
      961 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     1021 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     1081 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     1141 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     1201 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     1261 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     1321 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     1381 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     1441 gggcaaagga atagagtaga tgccgaccgg gatccactta acgttactga aatcatcaaa
     1501 cagcttgacg aatctggata taagatcgtt ggtgtcgatg tcagctccgg agttgagaca
     1561 aatggtgttc aggatctcga taagatacgt tcatttgtcc aagcagcaaa gagtgccttc
     1621 tagtgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt acctcttcca
     1681 gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt acgcccttca
     1741 ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg ggagacgaga
     1801 tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct cttggctcca
     1861 cgcgactata tatttgtctc taattgtact ttgacatgct cctcttcttt actctgatag
     1921 cttgactatg aaaattccgt caccagcccc tgggttcgca aagataattg cactgtttct
     1981 tccttgaact ctcaagccta caggacacac attcatcgta ggtataaacc tcgaaaatca
     2041 ttcctactaa gatgggtata caatagtaac catgcatggt tgcctagtga atgctccgta
     2101 acacccaata cgccggccga aactttttta caactctcct atgagtcgtt tacccagaat
     2161 gcacaggtac acttgtttag aggtaatcct tctttctaga agtcctcgtg tactgtgtaa
     2221 gcgcccactc cacatctcca ctcggttaac gctagcatgg atgttttccc agtcacgacg
     2281 ttgtaaaacg acggccagtc ttaagctcgg gccccaaata atgattttat tttgactgat
     2341 agtgacctgt tcgttgcaac aaattgatga gcaatgcttt tttataatgc caactttgta
     2401 caaaaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
     2461 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatgaatcaa
     2521 ctacttagat ggtattagtg acctgtagtc gaccgacagc cttccaaatg ttcttcgggt
     2581 gatgctgcca acttagtcga ccgacagcct tccaaatgtt cttctcaaac ggaatcgtcg
     2641 tatccagcct actcgctatt gtcctcaatg ccgtattaaa tcataaaaag aaataagaaa
     2701 aagaggtgcg agcctctttt ttgtgtgaca aaataaaaac atctacctat tcatatacgc
     2761 tagtgtcata gtcctgaaaa tcatctgcat caagaacaat ttcacaactc ttatactttt
     2821 ctcttacaag tcgttcggct tcatctggat tttcagcctc tatacttact aaacgtgata
     2881 aagtttctgt aatttctact gtatcgacct gcagactggc tgtgtataag ggagcctgac
     2941 atttatattc cccagaacat caggttaatg gcgtttttga tgtcattttc gcggtggctg
     3001 agatcagcca cttcttcccc gataacggag accggcacac tggccatatc ggtggtcatc
     3061 atgcgccagc tttcatcccc gatatgcacc accgggtaaa gttcacggga gactttatct
     3121 gacagcagac gtgcactggc cagggggatc accatccgtc gcccgggcgt gtcaataata
     3181 tcactctgta catccacaaa cagacgataa cggctctctc ttttataggt gtaaacctta
     3241 aactgcattt caccagcccc tgttctcgtc agcaaaagag ccgttcattt caataaaccg
     3301 ggcgacctca gccatccctt cctgattttc cgctttccag cgttcggcac gcagacgacg
     3361 ggcttcattc tgcatggttg tgcttaccag accggagata ttgacatcat atatgccttg
     3421 agcaactgat agctgtcgct gtcaactgtc actgtaatac gctgcttcat agcatacctc
     3481 tttttgacat acttcgggta tacatatcag tatatattct tataccgcaa aaatcagcgc
     3541 gcaaatacgc atactgttat ctggctttta gtaagccgga tccacgcggc gtttacgccc
     3601 cccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     3661 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     3721 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     3781 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     3841 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     3901 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     3961 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     4021 cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     4081 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     4141 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     4201 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     4261 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     4321 atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc
     4381 ccagggcttc ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc
     4441 cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tagtcgacta
     4501 caggtcacta ataccatcta agtagttgat tcatagtgac tggatatgtt gtgttttaca
     4561 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt
     4621 ttacgtttct cgttcagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
     4681 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     4741 tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc
     4801 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     4861 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     4921 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     4981 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     5041 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     5101 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     5161 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
     5221 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     5281 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     5341 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     5401 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     5461 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     5521 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     5581 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     5641 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     5701 gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg
     5761 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     5821 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5881 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5941 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6001 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6061 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6121 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6181 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6241 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6301 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6361 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6421 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6481 cttttgctgg ccttttgctc acatgtt
//
