LOCUS       sequence-56591-e        6972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6972)
  AUTHORS   Midelfort KS, Hernandez HH, Lippow SM, Tidor B, Drennan CL, Wittrup 
            KD
  TITLE     Substantial energetic improvement with minimal structural 
            perturbation in a high affinity mutant antibody.
  JOURNAL   J Mol Biol. 2004 Oct 22;343(3):685-701.
  PUBMED    15465055
REFERENCE   2  (bases 1 to 6972)
  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: "J Mol 
            Biol."; date: "2004-10-22"; volume: "343(3)"; pages: "685-701"
COMMENT     Sequence Label: pCT-4M5.3
FEATURES             Location/Qualifiers
     source          1..6972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(159..833)
                     /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(834..1114)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(1131..1150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1250..1272
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1310..1328)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    1370..1873
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        1910..2014
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2015..2083,2084..2875)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2233..2252)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3046..3634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3788..3805
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(3958..3963,3964..3981,3982..3988)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3996..4012
                     /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     4001..4023
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4020..4036
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4020..4036
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4057..4075
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(4243..4573)
                     /gene="S. cerevisiae GAL10"
                     /label=GAL10 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    4456..4573
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     CDS             5163..5174
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5175..5201
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6030..6059
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(6392..6411)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6393..6411)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6418..6435)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6418..6434)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6427..6449)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      join(6579..6972,1..62)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6666..6685)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6876..6897
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
       61 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caggcaagtg
      121 cacaaacaat acttaaataa atactactca gtaataacct atttcttagc atttttgacg
      181 aaatttgcta ttttgttaga gtcttttaca ccatttgtct ccacacctcc gcttacatca
      241 acaccaataa cgccatttaa tctaagcgca tcaccaacat tttctggcgt cagtccacca
      301 gctaacataa aatgtaagct ttcggggctc tcttgccttc caacccagtc agaaatcgag
      361 ttccaatcca aaagttcacc tgtcccacct gcttctgaat caaacaaggg aataaacgaa
      421 tgaggtttct gtgaagctgc actgagtagt atgttgcagt cttttggaaa tacgagtctt
      481 ttaataactg gcaaaccgag gaactcttgg tattcttgcc acgactcatc tccatgcagt
      541 tggacgatat caatgccgta atcattgacc agagccaaaa catcctcctt aggttgatta
      601 cgaaacacgc caaccaagta tttcggagtg cctgaactat ttttatatgc ttttacaaga
      661 cttgaaattt tccttgcaat aaccgggtca attgttctct ttctattggg cacacatata
      721 atacccagca agtcagcatc ggaatctaga gcacattctg cggcctctgt gctctgcaag
      781 ccgcaaactt tcaccaatgg accagaacta cctgtgaaat taataacaga catactccaa
      841 gctgcctttg tgtgcttaat cacgtatact cacgtgctca atagtcacca atgccctccc
      901 tcttggccct ctccttttct tttttcgacc gaattaattc ttaatcggca aaaaaagaaa
      961 agctccggat caagattgta cgtaaggtga caagctattt ttcaataaag aatatcttcc
     1021 actactgcca tctggcgtca taactgcaaa gtacacatat attacgatgc tgtctattaa
     1081 atgcttccta tattatatat atagtaatgt cgtttatggt gcactctcag tacaatctgc
     1141 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     1201 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     1261 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     1321 cgcctatttt tataggttaa tgtcatgata ataatggttt cttaggacgg atcgcttgcc
     1381 tgtaacttac acgcgcctcg tatcttttaa tgatggaata atttgggaat ttactctgtg
     1441 tttatttatt tttatgtttt gtatttggat tttagaaagt aaataaagaa ggtagaagag
     1501 ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa atttcaacaa aaagcgtact
     1561 ttacatatat atttattaga caagaaaagc agattaaata gatatacatt cgattaacga
     1621 taagtaaaat gtaaaatcac aggattttcg tgtgtggtct tctacacaga caagatgaaa
     1681 caattcggca ttaatacctg agagcaggaa gagcaagata aaaggtagta tttgttggcg
     1741 atccccctag agtcttttac atcttcggaa aacaaaaact attttttctt taatttcttt
     1801 ttttactttc tatttttaat ttatatattt atattaaaaa atttaaatta taattatttt
     1861 tatagcacgt gatgaaaagg acccaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1921 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1981 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2041 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2101 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2161 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2221 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2281 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2341 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2401 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2461 ttttcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2521 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     2581 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2641 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2701 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2761 agatggtaag ccctcccgta tcgtagttat ctacacgacg ggcagtcagg caactatgga
     2821 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2881 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2941 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3001 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3061 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3121 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3181 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3241 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3301 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3361 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3421 atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3481 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc caggggggaa
     3541 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3601 gtgatgctcg tcaggggggc cgagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3661 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3721 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3781 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     3841 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     3901 gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact cattaggcac cccaggcttt
     3961 acactttatg cttccggctc ctatgttgtg tggaattgtg agcggataac aatttcacac
     4021 aggaaacagc tatgaccatg attacgccaa gctcggaatt aaccctcact aaagggaaca
     4081 aaagctgggt acccgacagg ttatcagcaa caacacagtc atatccattc tcaattagct
     4141 ctaccacagt gtgtgaacca atgtatccag caccacctgt aaccaaaaca attttagaag
     4201 tactttcact ttgtaactga gctgtcattt atattgaatt ttcaaaaatt cttacttttt
     4261 ttttggatgg acgcaaagaa gtttaataat catattacat ggcattacca ccatatacat
     4321 atccatatac atatccatat ctaatcttac ttatatgttg tggaaatgta aagagcccca
     4381 ttatcttagc ctaaaaaaac cttctctttg gaactttcag taatacgctt aactgctcat
     4441 tgctatattg aagtacggat tagaagccgc cgagcgggtg acagccctcc gaaggaagac
     4501 tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg aaacgcagat gtgcctcgcg
     4561 ccgcactgct ccgaacaata aagattctac aatactagct tttatggtta tgaagaggaa
     4621 aaattggcag taacctggcc ccacaaacct tcaaatgaac gaatcaaatt aacaaccata
     4681 ggatgataat gcgattagtt ttttagcctt atttctgggg taattaatca gcgaagcgat
     4741 gatttttgat ctattaacag atatataaat gcaaaaactg cataaccact ttaactaata
     4801 ctttcaacat tttcggtttg tattacttct tattcaaatg taataaaaga tcgaattccc
     4861 tacttcatac attttcaatt aagatgcagt tacttcgctg tttttcaata ttttctgtta
     4921 ttgcttcagt tttagcacag gaactgacaa ctatatgcga gcaaatcccc tcaccaactt
     4981 tagaatcgac gccgtactct ttgtcaacga ctactatttt ggccaacggg aaggcaatgc
     5041 aaggagtttt tgaatattac aaatcagtaa cgtttgtcag taattgcggt tctcacccct
     5101 caacaactag caaaggcagc cccataaaca cacagtatgt ttttaaggac aatagctcga
     5161 cgattgaagg tagataccca tacgacgttc cagactacgc tctgcaggct agtggtggtg
     5221 gtggttctgg tggtggtggt tctggtggtg gtggttctgc tagcgacgtc gttatgactc
     5281 aaacaccact atcacttcct gttagtctag gtgatcaagc ctccatctct tgcagatcta
     5341 gtcagagcct cgtacacagt aatggaaaca cctatttacg ttggtacctg cagaagccag
     5401 gccagtctcc aaaggtcctg atctacaaag tttccaaccg agtttctggg gtcccagaca
     5461 ggttcagtgg cagtggatca gggacagatt tcacactcaa gatcaacaga gtggaggctg
     5521 aggatctggg agtttatttc tgctctcaaa gtacacatgt tccgtggacg ttcggtggag
     5581 gcaccaagct tgaaattaag tcctctgctg atgatgctaa gaaggatgct gctaagaagg
     5641 atgatgctaa gaaagatgat gctaagaaag atggtggcgt caaactggat gagactggag
     5701 gaggcttggt gcaacctggg ggggccatga aactctcctg tgttacctct ggattcactt
     5761 ttggtcacta ctggatgaac tgggtccgcc agtctccaga gaaaggactg gagtgggtag
     5821 cacaatttag aaacaaacct tataattatg aaacatatta ttcagattct gtgaaaggca
     5881 gattcaccat ctcaagagat gattccaaaa gtagtgtcta tctgcaaatg aacaacttaa
     5941 gagttgaaga cacgggtatc tattactgta cgggtgcttc ctatggtatg gaatacttgg
     6001 gtcaaggaac ctcagtcacc gtctcctcag aacaaaagct tatttctgaa gaagacttgt
     6061 aatagctcga gatctgataa caacagtgta gatgtaacaa aatcgacttt gttcccactg
     6121 tacttttagc tcgtacaaaa tacaatatac ttttcatttc tccgtaaaca acatgttttc
     6181 ccatgtaata tccttttcta tttttcgttc cgttaccaac tttacacata ctttatatag
     6241 ctattcactt ctatacacta aaaaactaag acaattttaa ttttgctgcc tgccatattt
     6301 caatttgtta taaattccta taatttatcc tattagtagc taaaaaaaga tgaatgtgaa
     6361 tcgaatccta agagaattga gctccaattc gccctatagt gagtcgtatt acaattcact
     6421 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     6481 tgcagcacat ccccccttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     6541 ttcccaacag ttgcgcagcc tgaatggcga atggcgcgac gcgccctgta gcggcgcatt
     6601 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     6661 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     6721 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6781 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6841 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6901 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6961 ctattggtta aa
//
