LOCUS       sequence-340017-        7036 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 7036)
  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 7036)
  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..7036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        319..983
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    535..652
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     947..970
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1301..1312
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1313..1339
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2168..2197
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2530..2549)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2531..2549)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2556..2573)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2556..2572)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2565..2587)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2713..3168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2800..2819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3010..3031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3271..3945)
                     /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(3946..4226)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4247..4266)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4366..4388
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4426..4444)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4485..4988
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5025..5129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5130..5198,5199..5990)
                     /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(5348..5367)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6161..6749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6650..6669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6903..6920
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag ctcgaaatta accctcacta aagggaacaa aagctggtac caattccttg
      181 aattttcaaa aattcttact ttttttttgg atggacgcaa agaagtttaa taatcatatt
      241 acatggcatt accaccatat acatatccat atacatatcc cactaaaggg aacaaaagct
      301 ggtaccaatt ccttgaattt tcaaaaattc ttactttttt tttggatgga cgcaaagaag
      361 tttaataatc atattacatg gcattaccac catatacata tccatataca tatccatatc
      421 taatcttact tatatgttgt ggaaatgtaa agagccccat tatcttagcc taaaaaaacc
      481 ttctctttgg aactttcagt aatacgctta actgctcatt gctatattga agtacggatt
      541 agaagccgcc gagcgggtga cagccctccg aaggaagact ctcctccgtg cgtcctcgtc
      601 ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc cgcactgctc cgaacaataa
      661 agattctaca atactagctt ttatggttat gaagaggaaa aattggcagt aacctggccc
      721 cacaaacctt caaatgaacg aatcaaatta acaaccatag gatgataatg cgattagttt
      781 tttagcctta tttctggggt aattaatcag cgaagcgatg atttttgatc tattaacaga
      841 tatataaatg caaaaactgc ataaccactt taactaatac tttcaacatt ttcggtttgt
      901 attacttctt attcaaatgt aataaaagta tcaacaaaaa attgttaata tacctctata
      961 ctttaacgtc aaggagaaaa accccggatc gaattcccta cttcatacat tttcaattaa
     1021 gatgcagtta cttcgctgtt tttcaatatt ttctgttatt gcttcagttt tagcacagga
     1081 actgacaact atatgcgagc aaatcccctc accaacttta gaatcgacgc cgtactcttt
     1141 gtcaacgact actattttgg ccaacgggaa ggcaatgcaa ggagtttttg aatattacaa
     1201 atcagtaacg tttgtcagta attgcggttc tcacccctca acaactagca aaggcagccc
     1261 cataaacaca cagtatgttt ttaaggacaa tagctcgacg attgaaggta gatacccata
     1321 cgacgttcca gactacgctc tgcaggctag tggtggtggt ggttctggtg gtggtggttc
     1381 tggtggtggt ggttctgcta gcgacgtcgt tatgactcaa acaccactat cacttcctgt
     1441 tagtctaggt gatcaagcct ccatctcttg cagatctagt cagagcctcg tacacagtaa
     1501 tggaaacacc tatttacgtt ggtacctgca gaagccaggc cagtctccaa aggtcctgat
     1561 ctacaaagtt tccaaccgag tttctggggt cccagacagg ttcagtggca gtggatcagg
     1621 gacagatttc acactcaaga tcaacagagt ggaggctgag gatctgggag tttatttctg
     1681 ctctcaaagt acacatgttc cgtggacgtt cggtggaggc accaagcttg aaattaagtc
     1741 ctctgctgat gatgctaaga aggatgctgc taagaaggat gatgctaaga aagatgatgc
     1801 taagaaagat ggtggcgtca aactggatga gactggagga ggcttggtgc aacctggggg
     1861 ggccatgaaa ctctcctgtg ttacctctgg attcactttt ggtcactact ggatgaactg
     1921 ggtccgccag tctccagaga aaggactgga gtgggtagca caatttagaa acaaacctta
     1981 taattatgaa acatattatt cagattctgt gaaaggcaga ttcaccatct caagagatga
     2041 ttccaaaagt agtgtctatc tgcaaatgaa caacttaaga gttgaagaca cgggtatcta
     2101 ttactgtacg ggtgcttcct atggtatgga atacttgggt caaggaacct cagtcaccgt
     2161 ctcctcagaa caaaagctta tttctgaaga agacttgtaa tagctcgaga tctgataaca
     2221 acagtgtaga tgtaacaaaa tcgactttgt tcccactgta cttttagctc gtacaaaata
     2281 caatatactt ttcatttctc cgtaaacaac atgttttccc atgtaatatc cttttctatt
     2341 tttcgttccg ttaccaactt tacacatact ttatatagct attcacttct atacactaaa
     2401 aaactaagac aattttaatt ttgctgcctg ccatatttca atttgttata aattcctata
     2461 atttatccta ttagtagcta aaaaaagatg aatgtgaatc gaatcctaag agaattgagc
     2521 tccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt
     2581 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2641 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2701 aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2761 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2821 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2881 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2941 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3001 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3061 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3121 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta
     3181 ttttctcctt acgcatctgt gcggtatttc acaccgcata gatcggcaag tgcacaaaca
     3241 atacttaaat aaatactact cagtaataac ctatttctta gcatttttga cgaaatttgc
     3301 tattttgtta gagtctttta caccatttgt ctccacacct ccgcttacat caacaccaat
     3361 aacgccattt aatctaagcg catcaccaac attttctggc gtcagtccac cagctaacat
     3421 aaaatgtaag ctttcggggc tctcttgcct tccaacccag tcagaaatcg agttccaatc
     3481 caaaagttca cctgtcccac ctgcttctga atcaaacaag ggaataaacg aatgaggttt
     3541 ctgtgaagct gcactgagta gtatgttgca gtcttttgga aatacgagtc ttttaataac
     3601 tggcaaaccg aggaactctt ggtattcttg ccacgactca tctccatgca gttggacgat
     3661 atcaatgccg taatcattga ccagagccaa aacatcctcc ttaggttgat tacgaaacac
     3721 gccaaccaag tatttcggag tgcctgaact atttttatat gcttttacaa gacttgaaat
     3781 tttccttgca ataaccgggt caattgttct ctttctattg ggcacacata taatacccag
     3841 caagtcagca tcggaatcta gagcacattc tgcggcctct gtgctctgca agccgcaaac
     3901 tttcaccaat ggaccagaac tacctgtgaa attaataaca gacatactcc aagctgcctt
     3961 tgtgtgctta atcacgtata ctcacgtgct caatagtcac caatgccctc cctcttggcc
     4021 ctctcctttt cttttttcga ccgaattaat tcttaatcgg caaaaaaaga aaagctccgg
     4081 atcaagattg tacgtaaggt gacaagctat ttttcaataa agaatatctt ccactactgc
     4141 catctggcgt cataactgca aagtacacat atattacgat gctgttctat taaatgcttc
     4201 ctatattata tatatagtaa tgtcgtgatc tatggtgcac tctcagtaca atctgctctg
     4261 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4321 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4381 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     4441 tatttttata ggttaatgtc atgataataa tggtttctta gacggatcgc ttgcctgtaa
     4501 cttacacgcg cctcgtatct tttaatgatg gaataatttg ggaatttact ctgtgtttat
     4561 ttatttttat gttttgtatt tggattttag aaagtaaata aagaaggtag aagagttacg
     4621 gaatgaagaa aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc gtactttaca
     4681 tatatattta ttagacaaga aaagcagatt aaatagatat acattcgatt aacgataagt
     4741 aaaatgtaaa atcacaggat tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt
     4801 cggcattaat acctgagagc aggaagagca agataaaagg tagtatttgt tggcgatccc
     4861 cctagagtct tttacatctt cggaaaacaa aaactatttt ttctttaatt tcttttttta
     4921 ctttctattt ttaatttata tatttatatt aaaaaattta aattataatt atttttatag
     4981 cacgtgatga aaaggaccca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5041 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5101 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5161 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5221 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5281 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5341 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5401 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5461 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5521 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5581 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5641 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5701 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5761 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5821 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5881 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5941 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6001 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6061 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6121 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6181 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6241 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6301 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6361 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6421 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6481 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6541 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6601 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6661 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6721 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6781 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6841 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6901 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     6961 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     7021 gtgagcgcaa cgcaat
//
