LOCUS       sequence-122726-        5499 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 5499)
  AUTHORS   Dantas G, Kuhlman B, Callender D, Wong M, Baker D
  TITLE     A large scale test of computational protein design: folding and 
            stability of nine completely redesigned globular proteins.
  JOURNAL   J Mol Biol. 2003 Sep 12. 332(2):449-60.
  PUBMED    12948494
REFERENCE   2  (bases 1 to 5499)
  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.
            2003 Sep 12. 332(2):449-60."
COMMENT     Sequence Label: PL2
FEATURES             Location/Qualifiers
     source          1..5499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(377..421)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     RBS             432..454
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    469..493
                     /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     complement(493..512)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(494..512)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(600..619)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        825..902
                     /gene="lacI"
                     /label=lacI promoter
     CDS             903..1985
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(922..941)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2794..2985
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2979..3001)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3087..3229
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3142..3161
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3244..3261)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3415..4003)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3495..3514)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4114..4133)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4125..4940
                     /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             4125..4940
                     /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(4198..4217)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5033..5488)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5170..5191)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5382..5401
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gaggcctgaa ccagagaata
      181 caattttaat aagttcgccg ccattttggt aatcgataga ataatcgccg tagttatcgc
      241 gtaagctttg tgcatattcc tgggcttgtt ttttcgctgc ttcctcactg cctgtaaatt
      301 cgattgtggt cgttttacca tcggcaaaga tgaaaatcac acgcacggta gtgtcgccgg
      361 aggtacccag atctgggctg tccatgtgct ggcgttcgaa tttagcagca gcggtttctt
      421 tcatatgtat atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc
      481 cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat cgatctcgat
      541 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
      601 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
      661 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
      721 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
      781 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
      841 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
      901 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
      961 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     1021 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     1081 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     1141 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     1201 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     1261 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     1321 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     1381 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     1441 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     1501 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     1561 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     1621 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     1681 ccgggctgcg cgttggtgcg gacatctcgg tagtgggata cgacgatacc gaagacagct
     1741 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     1801 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     1861 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     1921 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1981 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
     2041 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     2101 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     2161 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
     2221 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
     2281 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg
     2341 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     2401 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     2461 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     2521 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     2581 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     2641 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     2701 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     2761 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     2821 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     2881 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     2941 cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3001 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3061 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     3121 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat
     3181 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     3241 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3301 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3361 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3421 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3481 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3541 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3601 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3661 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3721 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3781 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3841 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3901 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3961 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4021 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa
     4081 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     4141 gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     4201 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     4261 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     4321 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     4381 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag
     4441 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     4501 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     4561 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     4621 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     4681 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     4741 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     4801 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     4861 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     4921 tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag
     4981 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgaaattgta
     5041 aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     5101 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     5161 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     5221 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     5281 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     5341 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     5401 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     5461 gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
