LOCUS       sequence-6765-ex        7360 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 7360)
  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 7360)
  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: pRK1043
FEATURES             Location/Qualifiers
     source          1..7360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /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             81..1163
                     /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(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    1176..1197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1528..2628
                     /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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2485..2494
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2602..2625
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2677..2688
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2689..3393
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="ESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKH
                     LFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQR
                     EERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIV
                     GIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEE
                     PFQPVKEATQLMN"
     primer_bind     complement(3450..3467)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3450..3466)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3459..3481)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3645..3662)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3815..3901
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3993..4020
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4040..4131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4132..4200,4201..4992)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4350..4369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5034..5547)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5229..5250)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5441..5460
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5658..6246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6147..6166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6400..6417
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6432..6572
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6498..6517)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6658..6680
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6674..6865)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagattatg aaaatcgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggcgaaag atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggga aagcttgttt
     2701 aaggggccgc gtgattacaa cccgatatcg agcaccattt gtcatttgac gaatgaatct
     2761 gatgggcaca caacatcgtt gtatggtatt ggatttggtc ccttcatcat tacaaacaag
     2821 cacttgttta gaagaaataa tggaacactg ttggtccaat cactacatgg tgtattcaag
     2881 gtcaagaaca ccacgacttt gcaacaacac ctcattgatg ggagggacat gataattatt
     2941 cgcatgccta aggatttccc accatttcct caaaagctga aatttagaga gccacaaagg
     3001 gaagagcgca tatgtcttgt gacaaccaac ttccaaacta agagcatgtc tagcatggtg
     3061 tcagacacta gttgcacatt cccttcatct gatggcatat tctggaagca ttggattcaa
     3121 accaaggatg ggcagtgtgg cagtccatta gtatcaacta gagatgggtt cattgttggt
     3181 atacactcag catcgaattt caccaacaca aacaattatt tcacaagcgt gccgaaaaac
     3241 ttcatggaat tgttgacaaa tcaggaggcg cagcagtggg ttagtggttg gcgattaaat
     3301 gctgactcag tattgtgggg gggccataaa gttttcatgg tgaaacctga agagcctttt
     3361 cagccagtta aggaagcgac tcaactcatg aatcgtcgtc gccgtcgcta ataaggatcc
     3421 tctagagtcg acctgcaggc aagcttggca ctggccgtcg ttttacaacg tcgtgactgg
     3481 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     3541 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3601 gaatggcagc ttggctgttt tggcggatga gataagattt tcagcctgat acagattaaa
     3661 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     3721 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     3781 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     3841 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     3901 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     3961 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     4021 tgcgtttcta caaactcttt ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4081 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4141 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4201 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4261 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4321 tctccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     4381 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4441 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4501 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4561 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4621 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4681 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4741 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4801 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4861 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4921 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4981 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaccccgg
     5041 ttgataatca gaaaagcccc aaaaacagga agattgtata agcaaatatt taaattgtaa
     5101 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     5161 aataggccga aatcggcaaa atcccttata aatcaaaaga atagcccgag atagggttga
     5221 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     5281 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt
     5341 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     5401 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     5461 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     5521 cgcttaatgc gccgctacag ggcgcgtaaa aggatctagg tgaagatcct ttttgataat
     5581 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5641 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5701 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5761 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     5821 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5881 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5941 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6001 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6061 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6121 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6181 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6241 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6301 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     6361 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     6421 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     6481 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     6541 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     6601 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     6661 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     6721 aaagctcatc agcgtggtcg tgcagcgatt cacagatgtc tgcctgttca tccgcgtcca
     6781 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     6841 gggcggtttt ttcctgtttg gtcacttgat gcctccgtgt aagggggaat ttctgttcat
     6901 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     6961 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     7021 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     7081 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     7141 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     7201 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     7261 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     7321 catgcgcacc cgtggccagg acccaacgct gcccgaaatt
//
