LOCUS       sequence-131396-        6676 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tumor Necrosis Factor Alpha Yeast Display Plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6676)
  AUTHORS   Kowalsky CA, Faber MS, Nath A, Dann HE, Kelly VW, Liu L, Shanker P, 
            Wagner EK, Maynard JA, Chan C, Whitehead TA
  TITLE     Rapid fine conformational epitope mapping using comprehensive 
            mutagenesis and deep sequencing.
  JOURNAL   J Biol Chem. 2015 Oct 30;290(44):26457-70. doi: 
            10.1074/jbc.M115.676635. Epub 2015 Aug 20.
  PUBMED    26296891
REFERENCE   2  (bases 1 to 6676)
  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"; doi: 
            "10.1074/jbc.M115.676635"; journalName: "J Biol Chem"; date: 
            "2015-10-30- 30"; volume: "290"; issue: "44"; pages: "26457-70"
COMMENT     Sequence Label: pETCON-TNF
FEATURES             Location/Qualifiers
     source          1..6676
                     /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             1739..1768
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2097..2116)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2098..2116)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2123..2140)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2123..2139)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2132..2154)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2280..2735
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2367..2386)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2577..2598
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2838..3512)
                     /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(3513..3793)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3814..3833)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3933..3955
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3993..4011)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4052..4555
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4592..4696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4697..4765,4766..5557)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4915..4934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5728..6316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6217..6236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6470..6487
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6604..6625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6640..6645,6646..6663,6664..6670)
                     /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 agccatatgg gcccccagag
     1201 ggaggagttc cccagggacc tgagcctgat cagccccctg gcccaggccg tgaggagcag
     1261 cagcaggacc cccagcgaca agcccgtggc ccacgtggtg gccaaccccc aggccgaggg
     1321 ccagctgcag tggctgaaca ggagggccaa cgccctgctg gccaacggcg tggagctgag
     1381 ggacaaccag ctggtggtgc ccagcgaggg cctgtacctg atctacagcc aggtgctgtt
     1441 caagggccag ggctgcccca gcacccacgt gctgctgacc cacaccatca gcaggatcgc
     1501 cgtgagctac cagaccaagg tgaacctgct gagcgccatc aagagcccct gccagaggga
     1561 gacccccgag ggcgccgagg ccaagccctg gtacgagccc atctacctgg gcggcgtgtt
     1621 ccagctggag aagggcgaca ggctgagcgc cgagatcaac aggcccgact acctggactt
     1681 cgccgagagc ggccaggtgt acttcggcat catcgccctg ctcgaggggg gcggatccga
     1741 acaaaagctt atttctgaag aggacttgta atagagatct gataacaaca gtgtagatgt
     1801 aacaaaatcg actttgttcc cactgtactt ttagctcgta caaaatacaa tatacttttc
     1861 atttctccgt aaacaacatg ttttcccatg taatatcctt ttctattttt cgttccgtta
     1921 ccaactttac acatacttta tatagctatt cacttctata cactaaaaaa ctaagacaat
     1981 tttaattttg ctgcctgcca tatttcaatt tgttataaat tcctataatt tatcctatta
     2041 gtagctaaaa aaagatgaat gtgaatcgaa tcctaagaga attgagctcc aattcgccct
     2101 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2161 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2221 gcgaagaggc ccgcaccgat cgcctttccc aacagttgcg cagcctgaat ggcgaatgga
     2281 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2341 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2401 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2461 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2521 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2581 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2641 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2701 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     2761 catctgtgcg gtatttcaca ccgcatagat cggcaagtgc acaaacaata cttaaataaa
     2821 tactactcag taataaccta tttcttagca tttttgacga aatttgctat tttgttagag
     2881 tcttttacac catttgtctc cacacctccg cttacatcaa caccaataac gccatttaat
     2941 ctaagcgcat caccaacatt ttctggcgtc agtccaccag ctaacataaa atgtaagctt
     3001 tcggggctct cttgccttcc aacccagtca gaaatcgagt tccaatccaa aagttcacct
     3061 gtcccacctg cttctgaatc aaacaaggga ataaacgaat gaggtttctg tgaagctgca
     3121 ctgagtagta tgttgcagtc ttttggaaat acgagtcttt taataactgg caaaccgagg
     3181 aactcttggt attcttgcca cgactcatct ccatgcagtt ggacgatatc aatgccgtaa
     3241 tcattgacca gagccaaaac atcctcctta ggttgattac gaaacacgcc aaccaagtat
     3301 ttcggagtgc ctgaactatt tttatatgct tttacaagac ttgaaatttt ccttgcaata
     3361 accgggtcaa ttgttctctt tctattgggc acacatataa tacccagcaa gtcagcatcg
     3421 gaatctagag cacattctgc ggcctctgtg ctctgcaagc cgcaaacttt caccaatgga
     3481 ccagaactac ctgtgaaatt aataacagac atactccaag ctgcctttgt gtgcttaatc
     3541 acgtatactc acgtgctcaa tagtcaccaa tgccctccct cttggccctc tccttttctt
     3601 ttttcgaccg aattaattct taatcggcaa aaaaagaaaa gctccggatc aagattgtac
     3661 gtaaggtgac aagctatttt tcaataaaga atatcttcca ctactgccat ctggcgtcat
     3721 aactgcaaag tacacatata ttacgatgct gttctattaa atgcttccta tattatatat
     3781 atagtaatgt cgtgatctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3841 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3901 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3961 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     4021 taatgtcatg ataataatgg tttcttagac ggatcgcttg cctgtaactt acacgcgcct
     4081 cgtatctttt aatgatggaa taatttggga atttactctg tgtttattta tttttatgtt
     4141 ttgtatttgg attttagaaa gtaaataaag aaggtagaag agttacggaa tgaagaaaaa
     4201 aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta ctttacatat atatttatta
     4261 gacaagaaaa gcagattaaa tagatataca ttcgattaac gataagtaaa atgtaaaatc
     4321 acaggatttt cgtgtgtggt cttctacaca gacaagatga aacaattcgg cattaatacc
     4381 tgagagcagg aagagcaaga taaaaggtag tatttgttgg cgatccccct agagtctttt
     4441 acatcttcgg aaaacaaaaa ctattttttc tttaatttct ttttttactt tctattttta
     4501 atttatatat ttatattaaa aaatttaaat tataattatt tttatagcac gtgatgaaaa
     4561 ggacccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4621 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4681 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4741 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4801 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4861 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4921 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4981 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5041 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5101 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5161 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5221 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5281 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5341 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5401 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5461 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5521 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5581 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5641 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5701 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5761 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5821 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5881 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5941 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6001 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6061 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6121 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6181 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6241 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6301 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6361 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     6421 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     6481 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     6541 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     6601 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     6661 tcgtatgttg tgtgga
//
