LOCUS       sequence-154221-        6898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pETconNK with TEM1(S70A,D179G) beta-lactamase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6898)
  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 6898)
  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-TEM1(S70A,D179G)
FEATURES             Location/Qualifiers
     source          1..6898
                     /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             1187..1978
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /label=bla(M)
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="MHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMM
                     ATFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAIT
                     MSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERGTTMPAAMA
                     TTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGII
                     AALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     primer_bind     complement(1339..1358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             1997..2026
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2355..2374)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2356..2374)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2381..2398)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2381..2397)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2390..2412)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2538..2993
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2625..2644)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2835..2856
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3096..3770)
                     /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(3771..4051)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4072..4091)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4191..4213
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4251..4269)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4310..4813
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4850..4954
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4955..5770
                     /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             4955..5770
                     /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(5028..5047)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5950..6538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6439..6458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6692..6709
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6826..6847
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6862..6867,6868..6885,6886..6892)
                     /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 acccagaaac
     1201 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1261 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1321 ggccactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     1381 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1441 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1501 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     1561 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     1621 gaatgaagcc ataccaaacg acgagcgtgg caccacgatg cctgcagcaa tggcaacaac
     1681 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     1741 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     1801 gtttattgct gataaatctg gagcaggtga gcgtgggtct cgcggtatca ttgcagcact
     1861 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     1921 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggct
     1981 cgaggggggc ggatccgaac aaaagcttat ttctgaagag gacttgtaat agagatctga
     2041 taacaacagt gtagatgtaa caaaatcgac tttgttccca ctgtactttt agctcgtaca
     2101 aaatacaata tacttttcat ttctccgtaa acaacatgtt ttcccatgta atatcctttt
     2161 ctatttttcg ttccgttacc aactttacac atactttata tagctattca cttctataca
     2221 ctaaaaaact aagacaattt taattttgct gcctgccata tttcaatttg ttataaattc
     2281 ctataattta tcctattagt agctaaaaaa agatgaatgt gaatcgaatc ctaagagaat
     2341 tgagctccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac
     2401 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     2461 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cctttcccaa cagttgcgca
     2521 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2581 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2641 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2701 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2761 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2821 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2881 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2941 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg
     3001 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagatcg gcaagtgcac
     3061 aaacaatact taaataaata ctactcagta ataacctatt tcttagcatt tttgacgaaa
     3121 tttgctattt tgttagagtc ttttacacca tttgtctcca cacctccgct tacatcaaca
     3181 ccaataacgc catttaatct aagcgcatca ccaacatttt ctggcgtcag tccaccagct
     3241 aacataaaat gtaagctttc ggggctctct tgccttccaa cccagtcaga aatcgagttc
     3301 caatccaaaa gttcacctgt cccacctgct tctgaatcaa acaagggaat aaacgaatga
     3361 ggtttctgtg aagctgcact gagtagtatg ttgcagtctt ttggaaatac gagtctttta
     3421 ataactggca aaccgaggaa ctcttggtat tcttgccacg actcatctcc atgcagttgg
     3481 acgatatcaa tgccgtaatc attgaccaga gccaaaacat cctccttagg ttgattacga
     3541 aacacgccaa ccaagtattt cggagtgcct gaactatttt tatatgcttt tacaagactt
     3601 gaaattttcc ttgcaataac cgggtcaatt gttctctttc tattgggcac acatataata
     3661 cccagcaagt cagcatcgga atctagagca cattctgcgg cctctgtgct ctgcaagccg
     3721 caaactttca ccaatggacc agaactacct gtgaaattaa taacagacat actccaagct
     3781 gcctttgtgt gcttaatcac gtatactcac gtgctcaata gtcaccaatg ccctccctct
     3841 tggccctctc cttttctttt ttcgaccgaa ttaattctta atcggcaaaa aaagaaaagc
     3901 tccggatcaa gattgtacgt aaggtgacaa gctatttttc aataaagaat atcttccact
     3961 actgccatct ggcgtcataa ctgcaaagta cacatatatt acgatgctgt tctattaaat
     4021 gcttcctata ttatatatat agtaatgtcg tgatctatgg tgcactctca gtacaatctg
     4081 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4141 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4201 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4261 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgg atcgcttgcc
     4321 tgtaacttac acgcgcctcg tatcttttaa tgatggaata atttgggaat ttactctgtg
     4381 tttatttatt tttatgtttt gtatttggat tttagaaagt aaataaagaa ggtagaagag
     4441 ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa atttcaacaa aaagcgtact
     4501 ttacatatat atttattaga caagaaaagc agattaaata gatatacatt cgattaacga
     4561 taagtaaaat gtaaaatcac aggattttcg tgtgtggtct tctacacaga caagatgaaa
     4621 caattcggca ttaatacctg agagcaggaa gagcaagata aaaggtagta tttgttggcg
     4681 atccccctag agtcttttac atcttcggaa aacaaaaact attttttctt taatttcttt
     4741 ttttactttc tatttttaat ttatatattt atattaaaaa atttaaatta taattatttt
     4801 tatagcacgt gatgaaaagg acccaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4861 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4921 ataaatgctt caataatatt gaaaaaggaa gagtatgagc catattcaac gggaaacgtc
     4981 ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     5041 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     5101 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     5161 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     5221 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     5281 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     5341 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     5401 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     5461 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     5521 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     5581 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     5641 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     5701 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     5761 gtttttctaa cctcagcaac tgtcagacca agtttactca tatatacttt agattgattt
     5821 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5881 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5941 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6001 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6061 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6121 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6181 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6241 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6301 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6361 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6421 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6481 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6541 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6601 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6661 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6721 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     6781 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     6841 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtgga
//
