LOCUS       Exported                8129 bp ds-DNA     circular SYN 22-DEC-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pVP65KR-gb-SacB(-)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8129)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, May 13, 2021 from SnapGene Viewer 5.2.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8129
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      20..106
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(139..849)
                     /codon_start=1
                     /product="tobacco vein mottling virus NIa protease 
                     (Nallamsetty et al., 2004)"
                     /label=TVMV protease
                     /translation="MSKALLKGVRDFNPISACVCLLENSSDGHSERLFGIGFGPYIIAN
                     QHLFRRNNGELTIKTMHGEFKVKNSTQLQMKPVEGRDIIVIKMAKDFPPFPQKLKFRQP
                     TIKDRVCMVSTNFQQKSVSSLVSESSHIVHKEDTSFWQHWITTKDGQCGSPLVSIIDGN
                     ILGIHSLTHTTNGSNYFVEFPEKFVATYLDAADGWCKNWKFNADKISWGSFTLVEDAPE
                     DDFMAKKTVAAIMD"
     RBS             855..863
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        complement(873..946)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    903..921
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    928..946
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        956..995
                     /label=gadAbolA
     primer_bind     976..1017
                     /label=bolAsensebinding
     RBS             1014..1025
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1032..2135
                     /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="MGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL
                     EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK
                     LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLI
                     AADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKG
                     ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF
                     LENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA
                     FWYAVRTAVINAASGRQTVDEALKDAQT"
     gene            2136..2846
                     /label=mCherry
     CDS             2847..2867
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     CDS             2868..2891
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2907..4325
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     protein_bind    complement(4473..4497)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      4583..4669
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4762..5844)
                     /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"
     promoter        complement(5845..5922)
                     /gene="lacI"
                     /label=lacI promoter
     misc_feature    6075..6215
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6401..6989)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7167..7961)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
ORIGIN
        1 cgtacgtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
       61 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctctagcacg
      121 cgtatcattg gatctttatt agtccatgat ggcggcaaca gtttttttgg ccatgaagtc
      181 atcttccggc gcatcttcaa ccagggtaaa ggaaccccag ctgattttat ccgcgttgaa
      241 tttccagttt ttgcaccaac catccgcggc atcgagataa gtcgccacga atttttccgg
      301 aaattccacg aagtagttgc taccgttggt ggtatgagtc aggctgtgga tgcccagaat
      361 gttgccatca atgatggaaa ctagtgggct gccacactgg ccatctttag tggtgatcca
      421 gtgctgccag aaagaagtgt cttctttatg cacaatgtga gaggattcag acaccaggct
      481 ggacacgctt ttctgctgaa agttggtgga caccatgcac acacgatctt tgatggtcgg
      541 ctgacggaat ttcagtttct gcgggaacgg cgggaagtct ttagccattt tgataacgat
      601 aatgtcacgg ccttcaaccg gtttcatctg cagctgggta gagtttttga ctttgaattc
      661 accgtgcatg gttttgatgg tcagttcgcc attgttacga cgaaacagat gctggttggc
      721 aatgatatac gggccaaaac caatgccaaa cagacgttca ctatgaccat ccgaggagtt
      781 ttccagcagg catacgcaag cagagatcgg attaaaatcg cgcacgccct tcagcaaagc
      841 cttagacata tcttacctcc ttaaatcaat tcggtcagtg cgtcctgctg atgtgctcag
      901 tatctctatc actgataggg atgtcaatct ctatcactga tagggactcg agaaagcctt
      961 gcttccattg cggatgtaaa agcggctagt atttatttca cacagaattc attaaagagg
     1021 agaaattaac catgggtaaa atcgaagaag gtaaactggt aatctggatt aacggcgata
     1081 aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag
     1141 tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg
     1201 atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc
     1261 tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg
     1321 atgcggtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc
     1381 tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc
     1441 tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt
     1501 acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca
     1561 agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc
     1621 tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag
     1681 ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca
     1741 tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat
     1801 ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc
     1861 tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata
     1921 aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc
     1981 cacgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc
     2041 agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc
     2101 agactgtcga tgaagccctg aaagacgcgc agactatggt ttccaagggc gaggaggata
     2161 acatggctat cattaaagag ttcatgcgct tcaaagttca catggagggt tctgttaacg
     2221 gtcacgagtt cgagatcgaa ggcgaaggcg agggccgtcc gtatgaaggc acccagaccg
     2281 ccaaactgaa agtgactaaa ggcggcccgc tgccttttgc gtgggacatc ctgagcccgc
     2341 aatttatgta cggttctaaa gcgtatgtta aacacccagc ggatatcccg gactatctga
     2401 agctgtcttt tccggaaggt ttcaagtggg aacgcgtaat gaattttgaa gatggtggtg
     2461 tcgtgaccgt cactcaggac tcctccctgc aggatggcga gttcatctat aaagttaaac
     2521 tgcgtggtac taattttcca tctgatggcc cggtgatgca gaaaaagacg atgggttggg
     2581 aggcgtctag cgaacgcatg tatccggaag atggtgcgct gaaaggcgaa attaaacagc
     2641 gcctgaaact gaaagatggc ggccattatg acgctgaagt gaaaaccacg tacaaagcca
     2701 agaaacctgt gcagctgcct ggcgcgtaca atgtgaatat taaactggac atcacctctc
     2761 ataatgaaga ttatacgatc gtagagcaat atgagcgcgc ggagggtcgt cattctaccg
     2821 gtggcatgga tgagctgtac aaaggggaaa ccgtgcgttt ccagtctcat caccatcatc
     2881 accatcacca tgcatccgcg atcgccatga acatcaaaaa gtttgcaaaa caagcaacag
     2941 tattaacctt tactaccgca ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa
     3001 cgaaccaaaa gccatataag gaaacatacg gcatttccca tattacacgc catgatatgc
     3061 tgcaaatccc tgaacagcaa aaaaatgaaa aatatcaagt tcctgaattc gattcgtcca
     3121 caattaaaaa tatctcttct gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa
     3181 acgctgacgg cactgtcgca aactatcacg gctaccacat cgtctttgca ttagccggag
     3241 atcctaaaaa tgcggatgac acatcgattt acatgttcta tcaaaaagtc ggcgaaactt
     3301 ctattgacag ctggaaaaac gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa
     3361 atgattctat cctaaaagac caaacacaag aatggtcagg ttcagccaca tttacatctg
     3421 acggaaaaat ccgtttattc tacactgatt tctccggtaa acattacggc aaacaaacac
     3481 tgacaactgc acaagttaac gtatcagcat cagacagctc tttgaacatc aacggtgtag
     3541 aggattataa atcaatcttt gacggtgacg gaaaaacgta tcaaaatgta cagcagttca
     3601 tcgatgaagg caactacagc tcaggcgaca accatacgct gagagatcct cactacgtag
     3661 aagataaagg ccacaaatac ttagtatttg aagcaaacac tggaactgaa gatggctacc
     3721 aaggcgaaga atctttattt aacaaagcat actatggcaa aagcacatca ttcttccgtc
     3781 aagaaagtca aaaacttctg caaagcgata aaaaacgcac ggctgagtta gcaaacggcg
     3841 ctctcggtat gattgagcta aacgatgatt acacactgaa aaaagtgatg aaaccgctga
     3901 ttgcatctaa cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca
     3961 aatggtacct gttcactgac tcccgcggat caaaaatgac gattgacggc attacgtcta
     4021 acgatattta catgcttggt tatgtttcta attctttaac tggcccatac aagccgctga
     4081 acaaaactgg ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc tttacttact
     4141 cacacttcgc tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa
     4201 acagaggatt ctacgcagac aaacaatcaa cgtttgcgcc gagcttcctg ctgaacatca
     4261 aaggcaagaa aacatctgtt gtcaaagaca gcatccttga acaaggacaa ttaacagtta
     4321 acaaattgtt taaacgaatt cgagctcggt acccggggat cctctagagt cgacctgcag
     4381 gcatgcaagc tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     4441 ccgctgagca ataactagca aactcgtttc tcgttcagct ttcttgtaca aagtggtgat
     4501 ggcgcgcctg taggacgtcg acggtaccat cgatacgcgt tcgaagcttc gcctggggta
     4561 atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     4621 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccctctag
     4681 attacgtgca gtcgatgata agctgtcaaa catgagaatt gtgcctaatg agtgagctaa
     4741 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4801 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4861 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4921 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4981 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5041 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5101 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5161 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5221 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5281 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5341 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5401 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5461 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5521 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5581 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5641 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5701 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5761 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5821 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5881 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tcggaaccta gagctgcctc
     5941 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     6001 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     6061 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
     6121 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
     6181 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     6241 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6301 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6361 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6421 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6481 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6541 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6601 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6661 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6721 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6781 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6841 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6901 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6961 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     7021 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     7081 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     7141 agtaaacttg gtctgacagc cctaggtcag aagaactcgt caagaaggcg atagaaggcg
     7201 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     7261 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     7321 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     7381 ggcaagcagg catcgccatg agtcacgacg agatcctcac catccggcat acgcgccttg
     7441 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     7501 tcgacaagac cggcttccat ccgagtgcgt gctcgctcga tgcgatgttt cgcttggtgg
     7561 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     7621 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     7681 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     7741 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     7801 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     7861 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     7921 gcggccggag aacctgcgtg caatccatct tgttcaagca tgcgaaacga ccgtcatcct
     7981 gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc
     8041 cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattctttt
     8101 gaagctcacg ctgccgcaag cactcaggg
//
