LOCUS       Exported                7408 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pRK792
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7408)
  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 7408)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7408
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        1406..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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     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             2692..2712
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2713..2733
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             2734..3441
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GKSLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMPKPE
                     EPFQPVKEATQLMN"
     primer_bind     complement(3498..3515)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3498..3514)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3507..3529)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3693..3710)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3863..3949
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4041..4068
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4088..4179
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4180..5040
                     /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(4398..4417)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5082..5595)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5277..5298)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5489..5508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5706..6294
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6195..6214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6448..6465
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6480..6620
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6546..6565)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6706..6728
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6722..6913)
                     /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 agggaagggg agaaaatctt
     2701 tattttcaag gtcatcatca tcatcatcat catggaaaaa gcttgtttaa ggggccgcgt
     2761 gattacaacc cgatatcgag caccatttgt catttgacga atgaatctga tgggcacaca
     2821 acatcgttgt atggtattgg atttggtccc ttcatcatta caaacaagca cttgtttaga
     2881 agaaataatg gaacactgtt ggtccaatca ctacatggtg tattcaaggt caagaacacc
     2941 acgactttgc aacaacacct cattgatggg agggacatga taattattcg catgcctaag
     3001 gatttcccac catttcctca aaagctgaaa tttagagagc cacaaaggga agaacgcata
     3061 tgtcttgtta caaccaactt ccaaactaag agcatgtcta gcatggtgtc agacactagt
     3121 tgcacattcc cttcatctga tggcatattc tggaagcatt ggattcaaac caaggatggg
     3181 cagtgtggca gtccattagt atcaactaga gatgggttca ttgttggtat acactcagca
     3241 tcgaatttca ccaacacaaa caattatttc acaagcgtgc cgaaaaactt catggaattg
     3301 ttgacaaatc aggaggcgca gcagtgggtt agtggttggc gattaaatgc tgactcagta
     3361 ttgtgggggg gccataaagt tttcatgccg aaacctgaag agccttttca gccagttaag
     3421 gaagcgactc aactcatgaa tcgtcgtcgc cgtcgctaat aaggatcctc tagagtcgac
     3481 ctgcaggcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     3541 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     3601 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcagctt
     3661 ggctgttttg gcggatgaga taagattttc agcctgatac agattaaatc agaacgcaga
     3721 agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc
     3781 atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg
     3841 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3901 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     3961 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     4021 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca
     4081 aactcttttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4141 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4201 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4261 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4321 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttc tccaatgatg
     4381 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     4441 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4501 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4561 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4621 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4681 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     4741 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4801 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4861 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4921 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4981 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5041 ctgtcagacc aagtttactc atatatactt tagattgatt taccccggtt gataatcaga
     5101 aaagccccaa aaacaggaag attgtataag caaatattta aattgtaaac gttaatattt
     5161 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     5221 tcggcaaaat cccttataaa tcaaaagaat agcccgagat agggttgagt gttgttccag
     5281 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     5341 tctatcaggg cgatggccca ctacgtgaac catcacccaa atcaagtttt ttggggtcga
     5401 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     5461 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5521 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5581 cgctacaggg cgcgtaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5641 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5701 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5761 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5821 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     5881 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5941 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6001 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6061 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6121 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6181 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6241 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6301 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6361 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     6421 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     6481 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     6541 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     6601 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     6661 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6721 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     6781 cgtggtcgtg cagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     6841 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     6901 cctgtttggt cacttgatgc ctccgtgtaa gggggaattt ctgttcatgg gggtaatgat
     6961 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     7021 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     7081 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     7141 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     7201 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     7261 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     7321 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     7381 tggccaggac ccaacgctgc ccgaaatt
//
