LOCUS pCT5-bac2.0 7877 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Cumate-inducible expression of sfGFP gene in Bacillus subtilis, B.
megaterium, and Escherichia coli.
ACCESSION .
VERSION .
KEYWORDS pCT5-bac2.0
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7877)
AUTHORS Seo SO, Schmidt-Dannert C
TITLE Development of a synthetic cumate-inducible gene expression system
for Bacillus.
JOURNAL Appl Microbiol Biotechnol. 2018 Nov 3. pii:
10.1007/s00253-018-9485-4. doi: 10.1007/s00253-018-9485-4.
PUBMED 30392122
REFERENCE 2 (bases 1 to 7877)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
//www.snapgene.com
FEATURES Location/Qualifiers
source 1..7877
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 14..41
/label=T7Te terminator
/note="phage T7 early transcription terminator"
CDS complement(77..688)
/codon_start=1
/gene="cymR from Pseudomonas putida"
/product="cumate repressor (Mullick et al., 2006)"
/label=CymR
/translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
protein_bind 1026..1053
/label=CuO
/bound_moiety="CymR"
/note="CymR-binding P2 operator sequence from the p-cmt
operon of Pseudomonas putida (Mullick et al., 2006)"
CDS 1082..1798
/codon_start=1
/product="GFP variant that folds robustly even when fused
to poorly folded proteins (Pedelacq et al., 2006)"
/label=superfolder GFP
/note="mammalian codon-optimized"
/translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
terminator 1849..1920
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
rep_origin complement(2101..2689)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2181..2200)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2860..3720)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 3483..3502
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(3721..3825)
/gene="bla"
/label=AmpR promoter
primer_bind 3893..3911
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
CDS complement(3989..5365)
/codon_start=1
/gene="Bacillus subtilis tetL"
/product="tetracycline efflux protein"
/label=TcR
/note="confers resistance to tetracycline"
/translation="MNTSYSQSNLRHNQILIWLCILSFFSVLNEMVLNVSLPDIANDFN
KPPASTNWVNTAFMLTFSIGTAVYGKLSDQLGIKRLLLFGIIINCFGSVIGFVGHSFFS
LLIMARFIQGAGAAAFPALVMVVVARYIPKENRGKAFGLIGSIVAMGEGVGPAIGGMIA
HYIHWSYLLLIPMITIITVPFLMKLLKKEVRIKGHFDIKGIILMSVGIVFFMLFTTSYS
ISFLIVSVLSFLIFVKHIRKVTDPFVDPGLGKNIPFMIGVLCGGIIFGTVAGFVSMVPY
MMKDVHQLSTAEIGSVIIFPGTMSVIIFGYIGGILVDRRGPLYVLNIGVTFLSVSFLTA
SFLLETTSWFMTIIIVFVLGGLSFTKTVISTIVSSSLKQQEAGAGMSLLNFTSFLSEGT
GIAIVGGLLSIPLLDQRLLPMEVDQSTYLYSNLLLLFSGIIVISWLVTLNVYKHSQRDF
"
CDS complement(5565..6569)
/codon_start=1
/gene="repB"
/product="RepB replication protein"
/label=repB
/note="from Enterococcus faecalis plasmid pAM-alpha-1"
/db_xref="GI:22652809"
/protein_id="AAN03827.1"
/translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
promoter 6994..7022
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
ORIGIN
1 actagttcac actggctcac cttcgggtgg gcctttctgc gtttatatac tagagagaga
61 atataaaaag ccattactag cgcttgaatt tcgcgtaccg ctctcgcgca atttcgagtg
121 tcgagttcct gacacgctca aagcgttctt tgtccttctg ccataggcta cgaacagcaa
181 gcccacgcac cgaattgaat atcaaccaaa ggatatcttc tgcatcatca cgcgaaagac
241 cacggctcac cagaacacca agccacatat cctcgacgac aaaccgattc ctctctaccg
301 tgcgctgaat accctcgcgt aacgctgggt cccggtcggc agccacaatc aaatcaaggc
361 tgatagagaa gtcatcgtcg aggaaaaatt cggcggcgtc gtccagcatt tgctggatga
421 cgtcatcctc tggcttcaat ttcgctaatc gagcccgact gcgttcggtg atctgttcgt
481 aaagccattc aaaagtggca agcagaagct caagctttgt cgggaaatga tggctctgcg
541 ctcctctgga gacaccagca gcaccgggca catctgcgat ccggaatccc gcgtaacctt
601 tttcccgtaa aacccccagg gccgctgcaa tcaacttgcc ctgggtctcc attgcgcgct
661 ctgcctgtgt tcttctcttt ggactcatga tcaccatttt ttttactaaa gcttgatctg
721 caatttgaat aataaccact cctttgttta tccaccgaac taagttggtg ttttttgaag
781 cttgaattag atatttaaaa gtatcatatc taatattata actaaatttt ctaaaaaaaa
841 cattgaagga acggttattt gattattaat ccggcttttt tattatttag gcaactgaaa
901 cgattcggat cctgtattac tattctattt tgtcaaaata attttattga caacgtctta
961 ttaacgttga tataatttaa attttatttg acaaaaatgg gctcgtgttg tacaataaat
1021 gtagtaacaa acagacaatc tggtctgttt gtattatgat taactaataa ggaggacaaa
1081 catgtcaaaa ggagaagaac tttttacagg tgtagtacct atcttggttg aattggatgg
1141 tgatgttaac ggtcacaaat tttctgtacg tggtgaaggt gaaggtgatg caactaacgg
1201 taaattgaca cttaaattca tttgtacaac tggaaaactt cctgttcctt ggcctactct
1261 tgttacaaca ttgacatatg gagtacaatg tttttcacgt tatcctgatc atatgaaacg
1321 tcacgatttt tttaaatctg ctatgccaga aggttatgta caagaacgta caatttcatt
1381 taaagatgac ggaacatata aaacacgtgc tgaagtaaaa ttcgaaggtg acactcttgt
1441 taatcgtatc gaattgaaag gaatcgattt caaagaagat ggtaacattt tgggacacaa
1501 acttgaatac aacttcaact ctcataatgt ttatatcaca gctgacaaac aaaaaaacgg
1561 tattaaagct aattttaaaa ttcgtcacaa tgttgaagat ggatctgttc aattggctga
1621 tcattatcaa caaaatacac caatcggaga cggaccagta ttgcttccag ataaccacta
1681 cctttctact caatcagttc tttcaaaaga tcctaacgaa aaacgtgacc atatggtact
1741 tcttgaattt gttacagcag caggtatcac tcacggtatg gacgaacttt ataaataagt
1801 taaaagtctc gtaaagcgtt ctatcaataa cccgttggtg ccaggcatca aataaaacga
1861 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
1921 tactagagga gctcatgccg gcggcacctc gctaacggat tcaccactcc aagaattgga
1981 gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc
2041 atcgcgtccg ccatctccag cagccgcacg cggcgcatct cgggccgcgt tgctggcgtt
2101 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
2161 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
2221 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
2281 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
2341 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
2401 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
2461 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
2521 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
2581 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
2641 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
2701 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
2761 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
2821 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
2881 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
2941 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
3001 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
3061 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
3121 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg
3181 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
3241 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
3301 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
3361 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
3421 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg
3481 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
3541 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
3601 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
3661 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
3721 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
3781 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
3841 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
3901 tatcacgagg ccctttcgtc ttcaagaatt cctgttataa aaaaaggatc aattttgaac
3961 tctctcccaa agttgatccc ttaacgattt agaaatccct ttgagaatgt ttatatacat
4021 tcaaggtaac cagccaacta atgacaatga ttcctgaaaa aagtaataac aaattactat
4081 acagataagt tgactgatca acttccatag gtaacaacct ttgatcaagt aagggtatgg
4141 ataataaacc acctacaatt gcaatacctg ttccctctga taaaaagctg gtaaagttaa
4201 gcaaactcat tccagcacca gcttcctgct gtttcaagct acttgaaaca attgttgata
4261 taactgtttt ggtgaacgaa agcccaccta aaacaaatac gattataatt gtcatgaacc
4321 atgatgttgt ttctaaaaga aaggaagcag ttaaaaagct aacagaaaga aatgtaactc
4381 cgatgtttaa cacgtataaa ggacctcttc tatcaacaag tatcccacca atgtagccga
4441 aaataatgac actcattgtt ccagggaaaa taattacact tccgatttcg gcagtactta
4501 gctggtgaac atctttcatc atataaggaa ccatagagac aaaccctgct actgttccaa
4561 atataattcc cccacaaaga actccaatca taaaaggtat atttttccct aatccgggat
4621 caacaaaagg atctgttact ttcctgatat gttttacaaa tatcaggaat gacagcacgc
4681 taacgataag aaaagaaatg ctatatgatg ttgtaaacaa cataaaaaat acaatgccta
4741 cagacattag tataattcct ttgatatcaa aatgaccttt tatccttact tctttcttta
4801 ataatttcat aagaaacgga acagtgataa ttgttatcat aggaatgagt agaagatagg
4861 accaatgaat ataatgggct atcattccac caatcgctgg accgactcct tctcccatgg
4921 ctactatcga tccaataaga ccaaatgctt tacccctatt ttcctttgga atatagcgcg
4981 caactacaac cattacgagt gctggaaatg cagctgcacc agccccttga ataaaacgag
5041 ccataataag taaggaaaag aaagaatggc caacaaaccc aattaccgac ccgaaacaat
5101 ttattataat tccaaatagg agtaaccttt tgatgcctaa ttgatcagat agctttccat
5161 atacagctgt tccaatggaa aaggttaaca taaaggctgt gttcacccag tttgtactcg
5221 caggtggttt attaaaatca tttgcaatat caggtaatga gacgttcaaa accatttcat
5281 ttaatacgct aaaaaaagat aaaatgcaaa gccaaattaa aatttggttg tgtcgtaaat
5341 tcgattgtga ataggatgta ttcacatttc accctccaat aatgagggca gacgtagttt
5401 atagggttaa tgatacgctt ccctctttta attgaaccct gttacattca ttacacttca
5461 taattaattc ctcctaaact tgattaaaac attttaccac atataaacta agttttaaat
5521 tcagtatttc atcacttata caacaatatg gcccgtttgt tgaactactc tttaataaaa
5581 taatttttcc gttcccaatt ccacattgca ataatagaaa atccatcttc atcggctttt
5641 tcgtcatcat ctgtatgaat caaatcgcct tcttctgtgt catcaaggtt taatttttta
5701 tgtatttctt ttaacaaacc accataggag attaaccttt tacggtgtaa accttcctcc
5761 aaatcagaca aacgtttcaa attcttttct tcatcatcgg tcataaaatc cgtatccttt
5821 acaggatatt ttgcagtttc gtcaattgcc gattgtatat ccgatttata tttatttttc
5881 ggtcgaatca tttgaacttt tacatttgga tcatagtcta atttcattgc ctttttccaa
5941 aattgaatcc attgtttttg attcacgtag ttttctgtat tcttaaaata agttggttcc
6001 acacatacca atacatgcat gtgctgatta taagaattat ctttattatt tattgtcact
6061 tccgttgcac gcataaaacc aacaagattt ttattaattt ttttatattg catcattcgg
6121 cgaaatcctt gagccatatc tgacaaactc ttatttaatt cttcgccatc ataaacattt
6181 ttaactgtta atgtgagaaa caaccaacga actgttggct tttgtttaat aacttcagca
6241 acaacctttt gtgactgaat gccatgtttc attgctctcc tccagttgca cattggacaa
6301 agcctggatt tacaaaacca cactcgatac aactttcttt cgcctgtttc acgattttgt
6361 ttatactcta atatttcagc acaatctttt actctttcag cctttttaaa ttcaagaata
6421 tgcagaagtt caaagtaatc aacattagcg attttctttt ctctccatgg tctcactttt
6481 ccactttttg tcttgtccac taaaaccctt gatttttcat ctgaataaat gctactatta
6541 ggacacataa tattaaaaga aacccccatc tatttagtta tttgtttggt cacttataac
6601 tttaacagat ggggtttttc tgtgcaacca attttaaggg ttttcaatac tttaaaacac
6661 atacatacca acacttcaac gcacctttca gcaactaaaa taaaaatgac gttatttcta
6721 tatgtatcaa gataagaaag aacaagttca aaaccatcaa aaaaagacac cttttcaggt
6781 gcttttttta ttttataaac tcattccctg atctcgactt cgttcttttt ttacctctcg
6841 gttatgagtt agttcaaatt cgttcttttt aggttctaaa tcgtgttttt cttggaattg
6901 tgctgtttta tcctttacct tgtctacaaa ccccttaaaa acgtttttaa aggcttttaa
6961 gccgtctgta cgttccttaa ggaattctca tgtttgacag cttatcatcg ataagcttta
7021 atgcggtagt ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa
7081 caatgcgctc atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt
7141 tatgccggta ctgccgggcc tcttgcggga tatcgtccat tccgacagca tcgccagtca
7201 ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg
7261 agcactgtcc gaccgctttg gccgccgccc agtcctgctc gcttcgctac ttggagccac
7321 tatcgactac gcgatcatgg cgaccacacc cgtcctgtgg atctgacgcg tgtaactgcg
7381 aaggtaaaac gggtgattga aaataaacta acaaatgaag aggaacgaaa gagaagtcta
7441 gaatttgtta cgtttatatc ggatgaatta ctgcaaaatg atacggagcc tcgcacattt
7501 attattcaga actctctttt atcgctagag aaaatccata ctctcgcaca gaccaaagag
7561 gtagaggaat ataaagaagt cataactact ctgacaagac atgattcata atttgaaaag
7621 caggtaaact aacccaaaag gcaagactct ccgtagttta gaaaagagca ggttgctaat
7681 aacatataaa cagccagttg ccgttatgat aggtgactgg ctgcatggga tgaaaaggtg
7741 agggtggaga cagacataac actcttaata gaagagggta attctttctc ttttatagaa
7801 aatcaattaa ttgaaagtag ctccttcatt cttaagatca aacgtgatat agtttgctaa
7861 cctttgcgtt cacttaa
//
