LOCUS       40924_28232        9089 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pLH334, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9089)
  AUTHORS   Xu Y.
  TITLE     Development of a markerless genetic manipulation system in 
            Aspergillus niger and its application in generating high organic 
            acid producing strains
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9089)
  AUTHORS   Xu Y.
  TITLE     Direct Submission
  JOURNAL   Submitted (01-FEB-2018) College of Biotechnology, Tianjin University
REFERENCE   3  (bases 1 to 9089)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9089)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (01-FEB-2018) College of Biotechnology, Tianjin University of 
            Science "
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..365
                     /label=trpC promoter
                     /note="promoter for Aspergillus nidulans trpC"
     CDS             371..742
                     /codon_start=1
                     /label=BleoR
                     /note="antibiotic-binding protein"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     protein_bind    770..803
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1174..2196
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANILFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     misc_recomb     2233..2266
                     /label=loxP
                     /note="loxP"
     protein_bind    complement(2233..2266)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(2306..2322)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2525..2549
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             3849..4475
                     /codon_start=1
                     /label=pVS1 StaA
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             4912..5976
                     /codon_start=1
                     /label=pVS1 RepA
                     /note="replication protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      6045..6239
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     misc_feature    6583..6723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6909..7497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7587..8378)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    8803..8827
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    complement(8905..9079)
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
ORIGIN
        1 ccgacagaag atgatattga aggagcattt tttgggcttg gctggagcta gtggaggtca
       61 acaatgaatg cctattttgg tttagtcgtc caggcggtga gcacaaaatt tgtgtcgttt
      121 gacaagatgg ttcatttagg caactggtca gatcagcccc acttgtagca gtagcggcgg
      181 cgctcgaagt gtgactctta ttagcagaca ggaacgagga cattattatc atctgctgct
      241 tggtgcacga taacttggtg cgtttgtcaa gcaaggtaag tggacgaccc ggtcatacct
      301 tcttaagttc gcccttcctc cctttatttc agattcaatc tgacttacct attctaccca
      361 agcatcgatc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
      421 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
      481 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
      541 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
      601 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
      661 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
      721 cttcgtggcc gaggagcagg actgaaggcc tgaattcgag ctcggatcca taacttcgta
      781 taatgtatgc tatacgaagt tatgtcgacg ttaactgata ttgaaggagc attttttggg
      841 cttggctgga gctagtggag gtcaacaatg aatgcctatt ttggtttagt cgtccaggcg
      901 gtgagcacaa aatttgtgtc gtttgacaag atggttcatt taggcaactg gtcagatcag
      961 ccccacttgt agcagtagcg gcggcgctcg aagtgtgact cttattagca gacaggaacg
     1021 aggacattat tatcatctgc tgcttggtgc acgataactt ggtgcgtttg tcaagcaagg
     1081 taagtggacg acccggtcat accttcttaa gttcgccctt cctcccttta tttcagattc
     1141 aatctgactt acctattcta cccaagcatc caaatgaaaa agcctgaact caccgcgacg
     1201 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     1261 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     1321 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     1381 gccgcgctcc cgattccgga agtgcttgac attggggagt tcagcgagag cctgacctat
     1441 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     1501 gctgttctcc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     1561 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     1621 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     1681 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     1741 gaagtccggc acctcgtgca tgcggatttc ggctccaaca atgtcctgac ggacaatggc
     1801 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     1861 gccaacatcc tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     1921 gagcggaggc atccggagct tgcaggatcg ccgcgcctcc gggcgtatat gctccgcatt
     1981 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     2041 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     2101 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     2161 aaccgacgcc ccagcactcg tccgagggca aaggaataga gtagatgccg accgggaacc
     2221 agttaacgtc gaataacttc gtataatgta tgctatacga agttattcta gaactagtgg
     2281 gcccctgcag gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     2341 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     2401 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     2461 gctagagcag cttgagcttg gatcagattg tcgtttcccg ccttcagttt aaactatcag
     2521 tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat tagaataacg
     2581 gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg catgccaacc
     2641 acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct atagtgcagt
     2701 cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca agtcctaagt
     2761 tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt gttttagtcg
     2821 cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca agagcgccgc
     2881 cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga ccaaccaacg
     2941 ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca ccggcaccag
     3001 gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg acgttgtgac
     3061 agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca ttgccgagcg
     3121 catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg acaccaccac
     3181 gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg agcgttccct
     3241 aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg
     3301 cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga tcgaccagga
     3361 aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga ccctgtaccg
     3421 cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg
     3481 tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac gccaagagga
     3541 acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac cgaagagatc
     3601 gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg
     3661 cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg gccggccagc
     3721 ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac
     3781 agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca aatacgcaag
     3841 gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc aagacgacca
     3901 tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg ttagtcgatt
     3961 ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg
     4021 ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc cggcgcgact
     4081 tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag
     4141 ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc accgccgacc
     4201 tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa gcggcctttg
     4261 tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag gcgctggccg
     4321 ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac ccaggcactg
     4381 ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc
     4441 aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta aagagaaaat
     4501 gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc
     4561 aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc agttgccggc
     4621 ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca ttaccgagct
     4681 gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa atgagtagat
     4741 gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc accgacgccg
     4801 tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc tgggttgtct
     4861 gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga cggtcgcaaa
     4921 ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga gaagttgaag
     4981 gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg tgaatcgtgg
     5041 caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg
     5101 tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc gatgctctat
     5161 gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag
     5221 cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca cgtagaggtt
     5281 tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact gatggcggtt
     5341 tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa gcccggccgc
     5401 gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga tggcggaaag
     5461 cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt tgccatgcag
     5521 cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga agccttgatt
     5581 agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga gatcgagcta
     5641 gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct gacggttcac
     5701 cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct ggcacgccgc
     5761 gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg cagtggcagc
     5821 gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg
     5881 ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt catgcgctac
     5941 cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca gatgctaggg
     6001 caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac
     6061 attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa cccaaagccg
     6121 tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa aggcgatttt
     6181 tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc ctgtgcataa
     6241 ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc
     6301 tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc aaaaatggct
     6361 ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc actcgaccgc
     6421 cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     6481 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     6541 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     6601 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     6661 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     6721 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     6781 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     6841 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6901 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6961 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     7021 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     7081 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     7141 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     7201 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     7261 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     7321 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     7381 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     7441 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     7501 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     7561 attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata atattttatt
     7621 ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata ctgttcttcc
     7681 ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt gtccgccctg
     7741 ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa gatgttgctg
     7801 tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt ctttaaaaaa
     7861 tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc gcaatccaca
     7921 tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc taagctattc
     7981 gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc cgcatacagc
     8041 tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag gacgccatcg
     8101 gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag gacctttgga
     8161 acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac atcataggtg
     8221 gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc tcccaccagc
     8281 ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta tttttcgatc
     8341 agttttttca attccggtga tattctcatt ttagccattt attatttcct tcctcttttc
     8401 tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc aattcactgt
     8461 tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg ttttcaaagt
     8521 tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca caggcagcaa
     8581 cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt gtttcaaacc
     8641 cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag tctgccgcct
     8701 tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat cgagtggtga
     8761 ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg
     8821 tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg
     8881 aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg gttttaggaa
     8941 ttagaaattt tattgataga agtattttac aaatacaaat acatactaag ggtttcttat
     9001 atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg ggaactactc
     9061 acacattatt atggagaaac tcgaggtac
//
