LOCUS       sequence-323557-        7405 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 7405)
  AUTHORS   Kapust RB, Tozser J, Fox JD, Anderson DE, Cherry S, Copeland TD, 
            Waugh DS
  TITLE     Tobacco etch virus protease: mechanism of autolysis and rational 
            design of stable mutants with wild-type catalytic proficiency.
  JOURNAL   Protein Eng. 2001 Dec . 14(12):993-1000.
  PUBMED    11809930
REFERENCE   2  (bases 1 to 7405)
  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: "Protein 
            Eng. 2001 Dec . 14(12):993-1000."
COMMENT     Sequence Label: pRK793
FEATURES             Location/Qualifiers
     source          1..7405
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(718..723,724..739,740..746)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    754..770
                     /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     759..781
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             840..1940
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1797..1806
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1914..1937
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1989..2000
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2004..2024
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2025..2045
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             2046..2753
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     primer_bind     complement(2810..2827)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2810..2826)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2819..2841)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3005..3022)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3175..3261
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3353..3380
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3399..3490
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3491..3559,3560..4351)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3709..3728)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4393..4906)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4588..4609)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4800..4819
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5017..5605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5506..5525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5759..5776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5791..5931
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5857..5876)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6017..6039
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6033..6224)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6720..6797
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             join(6798..7405,1..475)
                     /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(6817..6836)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
       61 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
      121 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
      181 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
      241 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
      301 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
      361 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      481 acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg acagcttatc
      541 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg
      601 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg
      661 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg
      721 acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag
      781 gaaacagcca gtccgtttag gtgttttcac gagcacttca ccaacaagga ccatagatta
      841 tgaaaactga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc
      901 tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc
      961 cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta
     1021 tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca
     1081 ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca
     1141 acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag
     1201 atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga
     1261 aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc
     1321 tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag
     1381 acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta
     1441 aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag
     1501 gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag
     1561 tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg
     1621 gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc
     1681 tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg
     1741 gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca
     1801 ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct
     1861 ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag
     1921 ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca
     1981 acctcgggat cgagggaagg ggagaaaatc tttattttca aggtcatcat catcatcatc
     2041 atcatggaga aagcttgttt aaggggccgc gtgattacaa cccgatatcg agcaccattt
     2101 gtcatttgac gaatgaatct gatgggcaca caacatcgtt gtatggtatt ggatttggtc
     2161 ccttcatcat tacaaacaag cacttgttta gaagaaataa tggaacactg ttggtccaat
     2221 cactacatgg tgtattcaag gtcaagaaca ccacgacttt gcaacaacac ctcattgatg
     2281 ggagggacat gataattatt cgcatgccta aggatttccc accatttcct caaaagctga
     2341 aatttagaga gccacaaagg gaagagcgca tatgtcttgt gacaaccaac ttccaaacta
     2401 agagcatgtc tagcatggtg tcagacacta gttgcacatt cccttcatct gatggcatat
     2461 tctggaagca ttggattcaa accaaggatg ggcagtgtgg cagtccatta gtatcaacta
     2521 gagatgggtt cattgttggt atacactcag catcgaattt caccaacaca aacaattatt
     2581 tcacaagcgt gccgaaaaac ttcatggaat tgttgacaaa tcaggaggcg cagcagtggg
     2641 ttagtggttg gcgattaaat gctgactcag tattgtgggg gggccataaa gttttcatgg
     2701 tgaaacctga agagcctttt cagccagtta aggaagcgac tcaactcatg aatcgtcgtc
     2761 gccgtcgcta ataaggatcc tctagagtcg acctgcaggc aagcttggca ctggccgtcg
     2821 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2881 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2941 agttgcgcag cctgaatggc gaatggcagc ttggctgttt tggcggatga gataagattt
     3001 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
     3061 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     3121 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     3181 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     3241 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     3301 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc
     3361 atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca
     3421 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3481 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3541 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3601 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3661 ttttcgcccc gaagaacgtt tcccaatgat gagcactttt aaagttctgc tatgtggcgc
     3721 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3781 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3841 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3901 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3961 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4021 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4081 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4141 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4201 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4261 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4321 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4381 ttagattgat ttaccccggt tgataatcag aaaagcccca aaaacaggaa gattgtataa
     4441 gcaaatattt aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     4501 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     4561 tagcccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     4621 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     4681 ccatcaccca aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     4741 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     4801 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     4861 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtaaaa ggatctaggt
     4921 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4981 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5041 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5101 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5161 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5221 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5281 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5341 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5401 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5461 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5521 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5581 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5641 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5701 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5761 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     5821 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     5881 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     5941 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6001 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6061 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gcagcgattc acagatgtct
     6121 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     6181 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     6241 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     6301 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     6361 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     6421 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     6481 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     6541 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     6601 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     6661 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgaaattccg
     6721 acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     6781 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     6841 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     6901 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     6961 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     7021 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     7081 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     7141 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     7201 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     7261 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     7321 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     7381 gtctgcgtct ggctggctgg cataa
//
