LOCUS       sequence-154220-        7423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pETconNK with levoglucosan kinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7423)
  AUTHORS   Klesmith JR, Bacik JP, Wrenbeck EE, Michalczyk R, Whitehead TA
  TITLE     Trade-offs between enzyme fitness and solubility illuminated by deep
            mutational scanning.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Feb 14. pii: 201614437. doi: 
            10.1073/pnas.1614437114.
  PUBMED    28196882
REFERENCE   2  (bases 1 to 7423)
  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: "Proc Natl 
            Acad Sci U S A. 2017 Feb 14. pii: 201614437. doi: 
            10.1073/pnas.1614437114."
COMMENT     Sequence Label: pETconNK-LGK
FEATURES             Location/Qualifiers
     source          1..7423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..18
                     /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     7..29
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     26..42
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     26..42
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        63..81
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        111..763
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    315..432
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     727..750
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1082..1093
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1094..1120
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2522..2551
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2880..2899)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2881..2899)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2906..2923)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2906..2922)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2915..2937)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3063..3518
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3150..3169)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3360..3381
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3621..4295)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(4296..4576)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4597..4616)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4716..4738
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4776..4794)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4835..5338
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5375..5479
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5480..6295
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5480..6295
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5553..5572)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6475..7063
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6964..6983
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7217..7234
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7351..7372
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7387..7392,7393..7410,7411..7417)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
       61 gaaattaacc ctcactaaag ggaacaaaag ctggtaccaa ttccttgaat tttcaaaaat
      121 tcttactttt tttttggatg gacgcaaaga agtttaataa tcatattaca tggcattacc
      181 accatataca tatccatatc taatcttact tatatgttgt ggaaatgtaa agagccccat
      241 tatcttagcc taaaaaaacc ttctctttgg aactttcagt aatacgctta actgctcatt
      301 gctatattga agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact
      361 ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc
      421 cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa
      481 aattggcagt aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag
      541 gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg
      601 atttttgatc tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac
      661 tttcaacatt ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa
      721 attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat cgaattccct
      781 acttcataca ttttcaatta agatgcagtt acttcgctgt ttttcaatat tttctgttat
      841 tgcttcagtt ttagcacagg aactgacaac tatatgcgag caaatcccct caccaacttt
      901 agaatcgacg ccgtactctt tgtcaacgac tactattttg gccaacggga aggcaatgca
      961 aggagttttt gaatattaca aatcagtaac gtttgtcagt aattgcggtt ctcacccctc
     1021 aacaactagc aaaggcagcc ccataaacac acagtatgtt tttaaggaca atagctcgac
     1081 gattgaaggt agatacccat acgacgttcc agactacgct ctgcaggcta gtggtggagg
     1141 aggctctggt ggaggcggta gcggaggcgg agggtcggct agccatatgc cgattgcgac
     1201 ctcaacgggt gataatgttc tggactttac ggttctgggc ctgaatagcg gtacgagtat
     1261 ggatggtatt gactgcgcac tgtgtcattt ctatcagaaa accccggatg ctccgatgga
     1321 atttgaactg ctggaatacg gcgaagttcc gctggcgcag ccgattaaac aacgtgtcat
     1381 gcgcatgatc ctggaagata ccacgagccc gtctgaactg tcagaagtca acgtgattct
     1441 gggtgaacat tttgcggatg ccgtccgtca gttcgcggcc gaacgcaatg tggatctgtc
     1501 aaccattgac gcaatcgctt cgcacggcca gacgatttgg ctgctgagta tgccggaaga
     1561 aggtcaagtg aaatccgccc tgaccatggc agaaggcgct atcctggcga gtcgtacggg
     1621 tattacctcc atcacggatt tccgtatttc cgaccaggca gctggtcgtc aaggtgcacc
     1681 gctgatcgca tttttcgatg ctctgctgct gcatcacccg accaaactgc gcgcgtgcca
     1741 gaacattggc ggtatcgcca atgtgtgttt tattccgccg gatgttgacg gccgtcgcac
     1801 cgatgaatat tacgattttg acacgggtcc gggcaacgtg ttcatcgacg cagtggttcg
     1861 tcattttacc aatggtgaac aggaatatga taaagacggt gctatgggca aacgcggtaa
     1921 agtcgatcag gaactggtgg atgactttct gaaaatgccg tatttccaac tggacccgcc
     1981 gaaaaccacg ggccgtgaag tttttcgcga taccctggca catgacctga ttcgtcgcgc
     2041 ggaagccaaa ggtctgagcc cggatgacat cgtggccacc acgacccgta ttacggcaca
     2101 ggctatcgtt gatcactatc gtcgctacgc gccgtcacaa gaaattgacg aaatcttcat
     2161 gtgcggcggt ggcgcctata acccgaatat tgtggaattt atccagcaat cgtacccgaa
     2221 caccaaaatt atgatgctgg atgaagcagg tgtcccggca ggtgcaaaag aagcgattac
     2281 gttcgcctgg cagggcatgg aagccctggt tggtcgtagc atcccggttc cgacccgtgt
     2341 cgaaacgcgc cagcactatg tgctgggcaa agttagcccg ggtctgaatt accgctctgt
     2401 gatgaaaaaa ggcatggcat ttggtggcga tgctcagcaa ctgccgtggg tttctgaaat
     2461 gatcgtgaag aaaaaaggca aagttatcac caacaactgg gcgctcgagg ggggcggatc
     2521 cgaacaaaag cttatttctg aagaggactt gtaatagaga tctgataaca acagtgtaga
     2581 tgtaacaaaa tcgactttgt tcccactgta cttttagctc gtacaaaata caatatactt
     2641 ttcatttctc cgtaaacaac atgttttccc atgtaatatc cttttctatt tttcgttccg
     2701 ttaccaactt tacacatact ttatatagct attcacttct atacactaaa aaactaagac
     2761 aattttaatt ttgctgcctg ccatatttca atttgttata aattcctata atttatccta
     2821 ttagtagcta aaaaaagatg aatgtgaatc gaatcctaag agaattgagc tccaattcgc
     2881 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     2941 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3001 atagcgaaga ggcccgcacc gatcgccttt cccaacagtt gcgcagcctg aatggcgaat
     3061 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3121 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3181 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3241 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3301 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3361 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3421 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3481 taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta ttttctcctt
     3541 acgcatctgt gcggtatttc acaccgcata gatcggcaag tgcacaaaca atacttaaat
     3601 aaatactact cagtaataac ctatttctta gcatttttga cgaaatttgc tattttgtta
     3661 gagtctttta caccatttgt ctccacacct ccgcttacat caacaccaat aacgccattt
     3721 aatctaagcg catcaccaac attttctggc gtcagtccac cagctaacat aaaatgtaag
     3781 ctttcggggc tctcttgcct tccaacccag tcagaaatcg agttccaatc caaaagttca
     3841 cctgtcccac ctgcttctga atcaaacaag ggaataaacg aatgaggttt ctgtgaagct
     3901 gcactgagta gtatgttgca gtcttttgga aatacgagtc ttttaataac tggcaaaccg
     3961 aggaactctt ggtattcttg ccacgactca tctccatgca gttggacgat atcaatgccg
     4021 taatcattga ccagagccaa aacatcctcc ttaggttgat tacgaaacac gccaaccaag
     4081 tatttcggag tgcctgaact atttttatat gcttttacaa gacttgaaat tttccttgca
     4141 ataaccgggt caattgttct ctttctattg ggcacacata taatacccag caagtcagca
     4201 tcggaatcta gagcacattc tgcggcctct gtgctctgca agccgcaaac tttcaccaat
     4261 ggaccagaac tacctgtgaa attaataaca gacatactcc aagctgcctt tgtgtgctta
     4321 atcacgtata ctcacgtgct caatagtcac caatgccctc cctcttggcc ctctcctttt
     4381 cttttttcga ccgaattaat tcttaatcgg caaaaaaaga aaagctccgg atcaagattg
     4441 tacgtaaggt gacaagctat ttttcaataa agaatatctt ccactactgc catctggcgt
     4501 cataactgca aagtacacat atattacgat gctgttctat taaatgcttc ctatattata
     4561 tatatagtaa tgtcgtgatc tatggtgcac tctcagtaca atctgctctg atgccgcata
     4621 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4681 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4741 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4801 ggttaatgtc atgataataa tggtttctta gacggatcgc ttgcctgtaa cttacacgcg
     4861 cctcgtatct tttaatgatg gaataatttg ggaatttact ctgtgtttat ttatttttat
     4921 gttttgtatt tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa
     4981 aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta
     5041 ttagacaaga aaagcagatt aaatagatat acattcgatt aacgataagt aaaatgtaaa
     5101 atcacaggat tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt cggcattaat
     5161 acctgagagc aggaagagca agataaaagg tagtatttgt tggcgatccc cctagagtct
     5221 tttacatctt cggaaaacaa aaactatttt ttctttaatt tcttttttta ctttctattt
     5281 ttaatttata tatttatatt aaaaaattta aattataatt atttttatag cacgtgatga
     5341 aaaggaccca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5401 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5461 atattgaaaa aggaagagta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     5521 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     5581 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     5641 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     5701 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     5761 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     5821 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     5881 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     5941 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     6001 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     6061 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     6121 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     6181 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     6241 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaacctca
     6301 gcaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6361 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6421 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6481 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6541 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6601 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6661 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6721 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6781 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6841 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6901 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6961 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7021 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7081 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7141 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7201 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     7261 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     7321 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     7381 ccaggcttta cactttatgc ttccggctcg tatgttgtgt gga
//
