LOCUS pML2424 9513 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Generation of unmarked gene deletions in Mycobacterium smegmatis.
ACCESSION .
VERSION .
KEYWORDS pML2424
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9513)
AUTHORS Ofer N, Wishkautzan M, Meijler M, Wang Y, Speer A, Niederweis M, Gur
E
TITLE Ectoine biosynthesis in Mycobacterium smegmatis.
JOURNAL Appl Environ Microbiol. 2012 Oct;78(20):7483-6. doi:
10.1128/AEM.01318-12. Epub 2012 Aug 10.
PUBMED 22885758
REFERENCE 2 (bases 1 to 9513)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..9513
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(149..168)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
promoter 206..638
/gene="sacR"
/label=sacB promoter
/note="sacB promoter and control region"
CDS 639..2060
/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"
rep_origin complement(2227..2815)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2307..2326)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
protein_bind 5937..5970
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
protein_bind 8378..8411
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 8796..9497
/codon_start=1
/product="dimeric variant of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=dTomato
/note="mammalian codon-optimized"
/translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
HLFLYGMDELYK"
primer_bind complement(8929..8947)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 9197..9216
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 9391..9414
/label=DsRed1-C
/note="DsRed1, forward primer"
ORIGIN
1 atctgattta ggatacattt ctagccacct gacgtcgggg ggggggggna aagccacgtt
61 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
121 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
181 acgtcttgct cgaccatata cctgccgttc actattattt agtgaaatga gatattatga
241 tattttctga attgtgatta aaaaggcaac tttatgccca tgcaacagaa actataaaaa
301 atacagagaa tgaaaagaaa cagatagatt ttttagttct ttaggcccgt agtctgcaaa
361 tccttttatg attttctatc aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag
421 agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca
481 agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg tcacccctta catattttag
541 gtcttttttt attgtgcgta actaacttgc catcttcaaa caggagggct ggaagaagca
601 gaccgctaac acagtacata aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa
661 aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt
721 ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac
781 gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa gttcctgaat
841 tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct
901 ggccattaca aaacgctgac ggcactgtcg caaactatca cggctaccac atcgtctttg
961 cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag
1021 tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca
1081 aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca
1141 catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg
1201 gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca
1261 tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg
1321 tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc
1381 ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg
1441 aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat
1501 cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt
1561 tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga
1621 tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta
1681 aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg
1741 gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat
1801 acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa
1861 cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa
1921 gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg ccaagcttcc
1981 tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac
2041 aattaacagt taacaaataa gcggccgctc gctgcgctcg gtcgttcggc tgcggcgagc
2101 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
2161 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
2221 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
2281 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
2341 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
2401 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
2461 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
2521 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
2581 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
2641 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
2701 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
2761 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
2821 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gagtatcgag ccgagaacgt
2881 tatcgaagtt agtcatgtgt aatcccctcg tttgaacttt ggattaagcg tagatacacc
2941 cttggacaag ccagttggat tcggagacaa gcaaattcag ccttaaaaag ggcgaggccc
3001 tgcggtggtg gaacaccgca gggcctctaa ccgctcgacg cgctgcacca accagcccgc
3061 gaacggctgg cagccagcgt aaggcgcggc tcatcgggcg gcgttcgcca cgatgtcctg
3121 cacttcgagc caagcctcga acacctgctg gtgtgcacga ctcacccggt tgttgacacc
3181 gcgcgcggcc gtgcgggctc ggtggggcgg ctctgtcgcc cttgccagcg tgagtagcgc
3241 gtacctcacc tcgcccaaca ggtcgcacac agccgattcg tacgccataa agccaggtga
3301 gcccaccagc tccgtaagtt cgggcgctgt gtggctcgta cccgcgcatt caggcggcag
3361 ggggtctaac gggtctaagg cggcgtgtac ggccgccaca gcggctctca gcggcccgga
3421 aacgtcctcg aaacgacgca tgtgttcctc ctggttggta caggtggttg ggggtgctcg
3481 gctgtcgctg gtgttccacc accagggctc gacgggagag cgggggagtg tgcagttgtg
3541 gggtggcccc tcagcgaaat atctgacttg gagctcgtgt cggaccatac accggtgatt
3601 aatcgtggtc tactaccaag cgtgagccac gtcgccgacg aatttgagca gctctggctg
3661 ccgtactggc cgctggcaag cgacgatctg ctcgagggga tctaccgcca aagccgcgcg
3721 tcggccctag gccgccggta catcgaggcg aacccaacag cgctggcaaa cctgctggtc
3781 gtggacgtag accatccaga cgcagcgctc cgagcgctca gcgcccgggg gtcccatccg
3841 ctgcccaacg cgatcgtggg caatcgcgcc aacggccacg cacacgcagt gtgggcactc
3901 aacgcccctg ttccacgcac cgaatacgcg cggcgtaagc cgctcgcata catggcggcg
3961 tgcgccgaag gccttcggcg cgccgtcgac ggcgaccgca gttactcagg cctcatgacc
4021 aaaaaccccg gccacatcgc ctgggaaacg gaatggctcc actcagatct ctacacactc
4081 agccacatcg aggccgagct cggcgcgaac atgccaccgc cgcgctggcg tcagcagacc
4141 acgtacaaag cggctccgac gccgctaggg cggaattgcg cactgttcga ttccgtcagg
4201 ttgtgggcct atcgtcccgc cctcatgcgg atctacctgc cgacccggaa cgtggacgga
4261 ctcggccgcg cgatctatgc cgagtgccac acgcgaaacg ccgaattctc gtgcaacgac
4321 gtgtgtcccg gaccgctacc ggacagcgag gtccgcgcca tcgccaacag catttggcgt
4381 tggatcacaa ccaagtcgcg catttgggcg gacgggatcg tggtctacga ggccacactc
4441 agtgcgcgcc agtcggccat ctcgcggaag ggcgcagcag cgcgcacggc ggcgagcaca
4501 gttgcgcggc gcgcaaagtc cgcgtcagcc atggaggcat tgctatgagc gacggctaca
4561 gcgacggcta caaccggcag ccgactgtcc gcaaaaagcg gcgcgtgacc gccgccgaag
4621 gcgctcgaat caccggacta tccgaacgcc acgtcgtccg gctcgtggcg caggaacgca
4681 gcgagtggct cgccgagcag gctgcacgcc gcgaacgcat ccgcgcctat cacgacgacg
4741 agggccactc ttggccgcaa acggccaaac atttcgggct gcatctggac accgttaagc
4801 gactcggcta tcgggcgagg aaagagcgtg cggcagaaca ggaagcggct caaaaggccc
4861 acaacgaagc cgacaatcca ccgctgttct aacgcaattg gggagcgggt gtcgcggggg
4921 ttccgtgggg ggttccgttg caacgggtcg gacaggtaaa agtcctggta gacgctagtt
4981 ttctggtttg ggccatgcct gtctcgttgc gtgtttcgtt gcgcccgttt tgaataccag
5041 ccagacgaga cggggttcta cgaatcttgg tcgataccaa gccatttccg ctgaatatcg
5101 gggagctcac cgccagaatc ggtggttgtg gtgatgtacg tggcgaactc cgttgtagtg
5161 cctgtggtgg catccgtggc cactctcgtt gcacggttcg ttgtgccgtt acaggccccg
5221 ttgacagctc accgaacgta gttaaaacat gctggtcaaa ctaggtttac caacgatacg
5281 agtcagctca tctagggcca gttctaggcg ttgttcgttg cgcggttcgt tgcgcatgtt
5341 tcgtgtggtt gctagatggc tccgcaacca cacgcttcga ggttgagtgc ttccagcacg
5401 ggcgcgatcc agaagaactt cgtcgtgcga ctgtcctcgt taacgctcga agcgatgccg
5461 atcaaatcgg ctgtgccgtc gcggttcttg cggtacagcg gcgcgcctga gtcgccatgt
5521 acggtgcgca gctgagcggt gatcgtgtac ttggcagcca caacgaccgg cccacaggtc
5581 tcgccggatt tcatcccgta cttgcagacc tcggtgccgt acggaacatc tttgcggtcc
5641 ttcagcaccg cacgcacggg gtatcttccg ccgattttga acgaagcgat cggtgttccc
5701 ggagcaaacg caatcacccc agcgtcggtg tagaccggca atagatcatc gtcgccggta
5761 atcgtggtgc cttcgtctct cgacgcaacg taggtaccca acgtccgctg tgtatccggt
5821 ttctctgcaa ccgaatacag caccgggccc cccctcgagt ttaaacattt ttctaaatac
5881 attcaaatat gtatccgctc atgagacaat aaccatgcat gtgctagctt taattaataa
5941 cttcgtatag catacattat acgaagttat acgcgttcaa gctttctaga gaataggaac
6001 ttcgagctcg aattcgataa aataaaaaag gggaccctag aggtcccctt ttttatttta
6061 tcgaattcgt tgtgacaatt taccgaacaa ctccgcggcc gggaagccga tctcggcttg
6121 aacgaattgt taggtggcgg tacttgggtc gatggccgcc gggggccggg ggcggcgccg
6181 ggcggcccgg ggcgtcaggc gccgggggcg gtgtccggcg gcccccagag gaactgcgcc
6241 agttcctccg gatcggtgaa gccggagaga tccagcgggg tctcctcgaa cacctcgaag
6301 tcgtgcagga aggtgaaggc gagcagttcg cgggcgaagt cctcggtccg cttccactgc
6361 gccccgtcga gcagcgcggc caggatctcg cggtcgcccc ggaaggcgtt gagatgcagt
6421 tgcaccaggc tgtagcggga gtctcccgca tagacgtcgg tgaagtcgac gatcccggtg
6481 acctcggtcg cggccaggtc cacgaagatg ttggtcccgt gcaggtcgcc gtggacgaac
6541 cggggttcgc ggccggccag cagcgtgtcc acgtccggca gccagtcctc caggcggtcc
6601 agcagccggg gcgagaggta gccccacccg cggtggtcct cgacggtcgc cgcgcggcgt
6661 tcccgcagca gttccgggaa gacctcggaa tggggggtga gcacggtgtt cccggtcagc
6721 ggcaccctgt gcagccggcc gagcacccgg ccgagttcgc gggccagggc gagcagcgcg
6781 ttccggtcgg tcgtgccgtc catcgcggac cgccaggtgg tgccggtcat ccggctcatc
6841 accaggtagg gccacggcca ggctccggtg ccgggccgca gctcgccgcg gccgaggagg
6901 cggggcaccg gcaccggggc gtccgccagg accgcgtacg cctccgactc cgacgcgagg
6961 ctctccggac cgcaccagtg ctcgccgaac agcttgatca ccgggccggg ctcgccgacc
7021 agtacggggt tggtgctctc gccgggcacc cgcagcaccg gcggcaccgg cagcccgagc
7081 tcctccaggg ctcggcgggc cagcggctcc cagaattcct ggtcgttccg caggctcgcg
7141 taggaatcat ccgaatcaat acggtcgaga agtaacaggg attcttgtgt cacagcggac
7201 ctctattcac agggtacggg ccggcttaat tccgcacggc cggtcgcgac acggcctgtc
7261 cgcaccgcgg atcaggcgtt gacgatgacg ggctggtgat aaatctactt gtacagctcg
7321 tccatgccgt gggtgatgcc ggcggcggtg acgaactcca gcaggaccat gtggtcgcgc
7381 ttctcgttcg ggtccttgga cagggcggac tgggtggaca ggtagtggtt gtccggcagc
7441 aggaccgggc cgtcgccgat cggggtgttc tgctggtagt ggtcggctag ctggacgcca
7501 ccgtcctcga tgttgtggcg ggtcttgaag ttggccttga tgccgttctt ctgcttgtcc
7561 gcggtgatgt agacgttgtg ggagttgtag ttgtactcca gcttgtggcc caggatgtta
7621 ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tgaccagggt gtcgccctcg
7681 aacttgacct cggcacgcgt cttgtagtta ccgtcgtcct tgaaggagat ggtgcgctcc
7741 tggacgtaac cctccggcat ggcggacttg aagaagtcgt ggcgcttcat gtggtccggg
7801 tagcgggaga agcactggac gccgtaggtc agggtggtga ccagggtcgg ccacgggacc
7861 ggcagcttac cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcgtcgccc
7921 tcaccctcgc cggagacgga gaacttgtgg ccgttgacgt caccgtccag ctcgaccagg
7981 atcgggacga cgccggtgaa cagctcctcg cccttcgaca ttattaacct cctttctgtt
8041 aagcatgcgg atcgtgctca tttcgggcgg cgaatctctc ggcgtcgaaa tgatgtatgc
8101 cgtgctggtc gtacacacgg gtactcccag agcctactcg accgagcaca cgcaggacaa
8161 actcgcccac cccgggtggg ttctcccgct cgtcagagac ccttctcaca ccgggcctgt
8221 gcgtgcgaac gcacagatca acgccgggat ggcgtagagc tcactgcgca gcaccgtggg
8281 gatgcggccg atcatcacgt cgcggatcgt gccgcctccc acggccgtga cggtgccgac
8341 gatccaatat ttctagagtt tatcttacag atctgggata acttcgtata gcatacatta
8401 tacgaagtta tttaaatctg gcgcgcctcg tatactcgac tgagcgtcag accccgtaga
8461 aaatatcaaa ggatcttctt ggtttaaact agtggatcaa tattggattg ccgtggtgca
8521 gggtctccca ttctcggaac ccggtgagcc gtcccatgtt cggggtttcc ctgccctcgt
8581 actcaccccg gaacgcgacg gcgtcggtcc agtagcaggt ctcggagcag tacgtcctcc
8641 actggccgtc gaccttctcg acgatctaga attggcacat ggcccgtcac aaggagttcg
8701 atccggacgt cgcactggac accgcgatgc gggtgttctg gcgcagcggc tacgcgcaca
8761 cgtcgaccga ggatcccaga aaggaggaat atataatggt gagcaagggc gaggaggtca
8821 tcaaagagtt catgcgcttc aaggtgcgca tggagggctc catgaacggc cacgagttcg
8881 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
8941 tgaccaaggg cggccccctg cccttcgcct gggacatcct gtccccccag ttcatgtacg
9001 gctccaaggc gtacgtgaag caccccgccg acatccccga ttacaagaag ctgtccttcc
9061 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggtctg gtgaccgtga
9121 cccaggactc ctccctgcag gacggcacgc tgatctacaa ggtgaagatg cgcggcacca
9181 acttcccccc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctccaccg
9241 agcgcctgta cccccgcgac ggcgtgctga agggcgagat ccaccaggcc ctgaagctga
9301 aggacggcgg ccactacctg gtggagttca agaccatcta catggccaag aagcccgtgc
9361 aactgcccgg ctactactac gtggacacca agctggacat cacctcccac aacgaggact
9421 acaccatcgt ggaacagtac gagcgctccg agggccgcca ccacctgttc ctgtacggca
9481 tggacgagct gtacaagtaa cccgggtacc gat
//
