LOCUS       sequence-177612-        5551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Complements mutations in Aspergillus nidulans gene lysB (AN5206) 
            (mutants require lysine).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5551)
  AUTHORS   Dohn JW Jr, Grubbs AW, Oakley CE, Oakley BR
  TITLE     New multi-marker strains and complementing genes for Aspergillus 
            nidulans molecular biology.
  JOURNAL   Fungal Genet Biol. 2018 Feb;111:1-6. doi: 10.1016/j.fgb.2018.01.003.
            Epub 2018 Jan 5.
  PUBMED    29309843
REFERENCE   2  (bases 1 to 5551)
  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: "Fungal 
            Genet Biol."; date: "2018-02"; volume: "111:1-6. doi"; pages: " 
            10.1016/j.fgb.2018.01.003. Epub 2018 Jan 5"
COMMENT     Sequence Label: pLO99
FEATURES             Location/Qualifiers
     source          1..5551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        133..151
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     133..150
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(223..234)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2333..2352)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2334..2352)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2359..2376)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2359..2375)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2368..2390)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2513..2815
                     /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="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2694..2713
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3164..3958
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3164..3958
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3218..3237)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3828..3847
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4158..4167
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4164..4538
                     /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"
     rep_origin      4676..5264
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5165..5184
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5418..5435
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag ctatttaggt gacactatag aatactcaag ctatgcatca agcttggtac
      181 cgagctcgga tccactagta acggccgcca gtgtgctgga attcgccctt caatgctctt
      241 caccctcttc tagcagatca atatagctcc cacagatgct tattgtttag acgatcggac
      301 aatattttaa cattcaatat acaacagaca ggtgagatac aattaaaatt ggatatattc
      361 gaactatcct atgtttggac ataaacagac ctttgaaata acaaccaaca ccagcattta
      421 agtgcactaa gtccagttgc tgctcattct acaaagtatt tctaatagat gagcttgtag
      481 aatctgtaag cagtctacac gtccatatat atactcctcc acaagactcc aagcttacat
      541 tcctttgcag gttgacggat cttacagggc agtccatggc atacgggctc agtcttcgca
      601 tgctggctag attatctgtt aagttatcag taccgaccaa gggcagttcc aatagcacgt
      661 ttattgtgtc caccataggg aatatacacg tactccgtag gtattccgta gatcagaagg
      721 acgaggcgaa caatggtaaa gacacacccg gctattaccg ccggagccac gttgatccgg
      781 gccgttggat ttcctcttgc tccgattgtc ggggagacct ttttctctgt ccagttcact
      841 cccagtcgac attattgcgc aatggctgct gctaggacgc ttcgcatagg tcaattggct
      901 ccttttccga tcaaagtaaa ctgtagctga cattctccca ttctaggtct catccccggt
      961 gacggtatcg gacgggaggt cattccggta tggagaacgt ccaaccccgg atttcctcca
     1021 catgacaatt gaaggtcaat gctaaccatg cggttcgtta ggctggtcgc cgtctgctgg
     1081 aatcccttcc ctcctctctc aacctgaagt tcagcttcgt tgacctggat gccggtttcg
     1141 acaccttcaa gcgcaccggt accgctctcc ctgacaagac cgttgagatc ttgaaaaagg
     1201 agtgcgatgg agctcttttc ggagccgtca ggtaaagtga aaccatactg attgtcttct
     1261 gcattctaaa cgtgtcatct ccagctcccc tagcaccaag gtcgctggct actcctcccc
     1321 cattgtcgct ctccgcaaga agctcgacct gtacgccaac gtccgtccag tcaagactac
     1381 cagcggcgac agcaagggca accctatcga tctggtcatc gtccgggaga acactgagga
     1441 cctctatgtg aaggaggagc agaccaagga cacccccaac ggcaaggtgg ccgaggccat
     1501 caagcgcatc tccgagaacg cttcctcccg catctccacc atcgctggtg agatcgctct
     1561 gcgccgtcag aagatccgcg acgttgccgc cacccccggc ctccgctcca aacccatggt
     1621 tactatcacc cacaagtcca acgttctgtc gcagaccgac ggtctcttcc gtgagaccgc
     1681 ccgcaaggcc ctctccgccg accgcttctc ctccgtcgag gtcgaggagc agatcgtcga
     1741 ctccatggtc tacaagctct tccgtcagcc cgagtactat gatgtgattg tcgcccccaa
     1801 tctctacggt gacattctgt ccgacggcgc tgccgccctg gtcggcagtc tgggtctggt
     1861 gcccagcgcc aacgtcggtg acggctttgc catcggtgag ccttgccacg gctccgcccc
     1921 cgacatcgag ggcaagggca ttgccaaccc catcgctacc ctgcgcagtg tcgccctgat
     1981 gctggagttc cttggcgaag agaacgccgc cgccaagatc tacaccgctg tcgatgccaa
     2041 cctcgacgag ggcaagtacc tctcccctga catgggcgga aaggctacca cccagcaggt
     2101 tctcgacgat gtcctgaaga gactttagag gtcgttacgt aaaagcggat gtcttattgc
     2161 gaactgtgtg atgcttgtat agagcgactg tatatctaaa taaaccatga ctacctgcgt
     2221 cttgaattct tcccatctct ccatagtgcc tcctctcaga cagaagggcg aattctgcag
     2281 atatccatca cactggcggc cgctcgagca tgcatctaga gggcccaatt cgccctatag
     2341 tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     2401 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     2461 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctatacgtac ggcagtttaa
     2521 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
     2581 tattgacacg ccggggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
     2641 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat
     2701 gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag
     2761 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc
     2821 aggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga aagccagtcc
     2881 gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga caagggaaaa
     2941 cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg
     3001 ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt
     3061 tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag
     3121 gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3181 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3241 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3301 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     3361 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3421 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3481 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3541 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3601 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3661 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     3721 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3781 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3841 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3901 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat
     3961 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4021 acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4081 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4141 aatagcacgt gaggagggcc accatggcca agttgaccag tgccgttccg gtgctcaccg
     4201 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     4261 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     4321 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     4381 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     4441 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     4501 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctaaaa cttcattttt
     4561 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4621 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4681 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4741 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4801 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4861 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4921 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4981 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5041 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5101 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5161 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5221 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5281 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5341 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5401 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     5461 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5521 actggaaagc gggcagtgag cgcaacgcaa t
//
