LOCUS       sequence-154219-        6137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pETconNK Empty Backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6137)
  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 6137)
  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
FEATURES             Location/Qualifiers
     source          1..6137
                     /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             1236..1265
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1594..1613)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1595..1613)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1620..1637)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1620..1636)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1629..1651)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1777..2232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1864..1883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2074..2095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2335..3009)
                     /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(3010..3290)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3311..3330)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3430..3452
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3490..3508)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3549..4052
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4089..4193
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4194..5009
                     /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             4194..5009
                     /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(4267..4286)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5189..5777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5678..5697
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5931..5948
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6065..6086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6101..6106,6107..6124,6125..6131)
                     /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 agccatatgt gaagcatgct
     1201 taattaaggc gccttatctc gaggggggcg gatccgaaca aaagcttatt tctgaagagg
     1261 acttgtaata gagatctgat aacaacagtg tagatgtaac aaaatcgact ttgttcccac
     1321 tgtactttta gctcgtacaa aatacaatat acttttcatt tctccgtaaa caacatgttt
     1381 tcccatgtaa tatccttttc tatttttcgt tccgttacca actttacaca tactttatat
     1441 agctattcac ttctatacac taaaaaacta agacaatttt aattttgctg cctgccatat
     1501 ttcaatttgt tataaattcc tataatttat cctattagta gctaaaaaaa gatgaatgtg
     1561 aatcgaatcc taagagaatt gagctccaat tcgccctata gtgagtcgta ttacaattca
     1621 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     1681 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     1741 ctttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
     1801 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1861 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1921 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1981 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2041 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2101 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2161 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2221 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2281 catagatcgg caagtgcaca aacaatactt aaataaatac tactcagtaa taacctattt
     2341 cttagcattt ttgacgaaat ttgctatttt gttagagtct tttacaccat ttgtctccac
     2401 acctccgctt acatcaacac caataacgcc atttaatcta agcgcatcac caacattttc
     2461 tggcgtcagt ccaccagcta acataaaatg taagctttcg gggctctctt gccttccaac
     2521 ccagtcagaa atcgagttcc aatccaaaag ttcacctgtc ccacctgctt ctgaatcaaa
     2581 caagggaata aacgaatgag gtttctgtga agctgcactg agtagtatgt tgcagtcttt
     2641 tggaaatacg agtcttttaa taactggcaa accgaggaac tcttggtatt cttgccacga
     2701 ctcatctcca tgcagttgga cgatatcaat gccgtaatca ttgaccagag ccaaaacatc
     2761 ctccttaggt tgattacgaa acacgccaac caagtatttc ggagtgcctg aactattttt
     2821 atatgctttt acaagacttg aaattttcct tgcaataacc gggtcaattg ttctctttct
     2881 attgggcaca catataatac ccagcaagtc agcatcggaa tctagagcac attctgcggc
     2941 ctctgtgctc tgcaagccgc aaactttcac caatggacca gaactacctg tgaaattaat
     3001 aacagacata ctccaagctg cctttgtgtg cttaatcacg tatactcacg tgctcaatag
     3061 tcaccaatgc cctccctctt ggccctctcc ttttcttttt tcgaccgaat taattcttaa
     3121 tcggcaaaaa aagaaaagct ccggatcaag attgtacgta aggtgacaag ctatttttca
     3181 ataaagaata tcttccacta ctgccatctg gcgtcataac tgcaaagtac acatatatta
     3241 cgatgctgtt ctattaaatg cttcctatat tatatatata gtaatgtcgt gatctatggt
     3301 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3361 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3421 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3481 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3541 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
     3601 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
     3661 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
     3721 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
     3781 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
     3841 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
     3901 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
     3961 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
     4021 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
     4081 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4141 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagcc
     4201 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     4261 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     4321 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     4381 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     4441 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     4501 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     4561 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     4621 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     4681 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     4741 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     4801 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     4861 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     4921 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     4981 ttcatttgat gctcgatgag tttttctaac ctcagcaact gtcagaccaa gtttactcat
     5041 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5101 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5161 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5221 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5281 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5341 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5401 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5461 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5521 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5581 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5641 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5701 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5761 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5821 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5881 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5941 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     6001 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     6061 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     6121 ctcgtatgtt gtgtgga
//
