LOCUS pCRISPRx-Sth1Cas9-L5 8881 bp DNA circular SYN 25-AUG-2024
DEFINITION Sth1Cas9, TetR, KanR, L5Int, attP to create frameshifts and gene
deletion in Mycobacteria.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8881)
AUTHORS Meijers AS, Troost R, Ummels R, Maaskant J, Speer A, Nejentsev S,
Bitter W, Kuijl CP
TITLE Efficient genome editing in pathogenic mycobacteria using
Streptococcus thermophilus CRISPR1-Cas9.
JOURNAL Tuberculosis (Edinb). 2020 Sep;124:101983. doi:
10.1016/j.tube.2020.101983. Epub 2020 Aug 12.
PUBMED 32829077
REFERENCE 2 (bases 1 to 8881)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 25, 2024 from SnapGene Server 7.0.3
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Tuberculosis (Edinb)."; date: "2020-09"; volume: "124:101983. doi";
pages: " 10.1016/j.tube.2020.101983. Epub 2020 Aug 12"
FEATURES Location/Qualifiers
source 1..8881
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(499..1087)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(579..598)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
protein_bind 1270..1288
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 1300..1318
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
terminator 4798..4825
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
CDS 5215..5838
/codon_start=1
/gene="tetR from transposon Tn10"
/product="tetracycline repressor TetR"
/label=TetR
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
primer_bind complement(5288..5307)
/label=Tet-R
/note="Tetracycline resistance gene, reverse primer"
primer_bind complement(7841..7860)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 7852..8667
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind complement(7925..7944)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
ORIGIN
1 atcaataaac gaaaggctca gtcgaaagac tgggcctttc gtttatctgt tgtttgaaaa
61 aaaaaagcgc cgcaactgcg gcgctttttt ttttgaattc tctgaccagg gaaaatagcc
121 ctctgacctg gggatttgcg atccctatca gtgatagata taatctggga ggagacgatt
181 aatgcgtctc ggtttttgta ctcgaaagaa gctacaaaga taaggcttca tgccgaaatc
241 aacaccctgt cattttatgg cagggtgttt tttttttgtc gacttgggga ccctagaggt
301 cccctttttt atttttttgg atgaagagca ttatcaatgg ctcttcacag gaagcttaaa
361 aaaaaaagcc ccgcgattgc ggggcttttt ttttttaata cggttatcca cagaatcagg
421 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
481 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
541 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
601 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
661 ctttctcccc tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
721 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
781 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
841 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
901 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
961 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
1021 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
1081 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcatggaa caataaacga
1141 aaggctcagt cgaaagactg ggcctttcgt ttatctgttg tttgaaaaaa aaaagccccg
1201 cgattgcggg gctttttttt ttcatatgtc tgaccaggga aaatagccct ctgacctggg
1261 gatttgactt ccctatcagt gatagagata atctgggagt ccctatcagt gatagagaag
1321 gcggtatcga tatgtcggac ctggtgctgg gcctggacat cggcatcggc tcggtgggcg
1381 tgggcatcct gaacaaggtg accggcgaga tcatccacaa gaactcgcgc atcttcccgg
1441 ccgcccaggc cgagaacaac ctggtgcgcc gcaccaaccg ccagggccgc cgcctggccc
1501 gccgcaagaa gcaccgccgc gtgcgcctga accgcctgtt cgaggagtcg ggcctgatca
1561 ccgacttcac caagatctcg atcaacctga acccgtacca gctgcgcgtg aagggcctga
1621 ccgacgagct gtcgaacgag gagctgttca tcgccctgaa gaacatggtg aagcaccgcg
1681 gcatctcgta cctggacgac gcctcggacg acggcaactc gtcggtgggc gactacgccc
1741 agatcgtgaa ggagaactcg aagcagctgg agaccaagac cccgggccag atccagctgg
1801 agcgctacca gacctacggc cagctgcgcg gcgacttcac cgtggagaag gacggcaaga
1861 agcaccgcct gatcaacgtg ttcccgacct cggcctaccg ctcggaggcc ctgcgcatcc
1921 tgcagaccca gcaggagttc aacccgcaga tcaccgacga gttcatcaac cgctacctgg
1981 agatcctgac cggcaagcgc aagtactacc acggcccggg caacgagaag tcgcgcaccg
2041 actacggccg ctaccgcacc tcgggcgaga ccctggacaa catcttcggc atcctgatcg
2101 gcaagtgcac cttctacccg gacgagttcc gcgccgccaa ggcctcgtac accgcccagg
2161 agttcaacct gctgaacgac ctgaacaacc tgaccgtgcc gaccgagacc aagaagctgt
2221 cgaaggagca gaagaaccag atcatcaact acgtgaagaa cgagaaggcc atgggcccgg
2281 ccaagctgtt caagtacatc gccaagctgc tgtcgtgcga cgtggccgac atcaagggct
2341 accgcatcga caagtcgggc aaggccgaga tccacacctt cgaggcctac cgcaagatga
2401 agaccctgga gaccctggac atcgagcaga tggaccgcga gaccctggac aagctggcct
2461 acgtgctgac cctgaacacc gagcgcgagg gcatccagga ggccctggag cacgagttcg
2521 ccgacggctc gttctcgcag aagcaggtgg acgagctggt gcagttccgc aaggccaact
2581 cgtcgatctt cggcaagggc tggcacaact tctcggtgaa gctgatgatg gagctgatcc
2641 cggagctgta cgagacctcg gaggagcaga tgaccatcct gacccgcctg ggcaagcaga
2701 agaccacctc gtcgtcgaac aagaccaagt acatcgacga gaagctgctg accgaggaga
2761 tctacaaccc ggtggtggcc aagtcggtgc gccaggccat caagatcgtg aacgccgcca
2821 tcaaggagta cggcgacttc gacaacatcg tgatcgagat ggcccgcgag accaacgagg
2881 acgacgagaa gaaggccatc cagaagatcc agaaggccaa caaggacgag aaggacgccg
2941 ccatgctgaa ggccgccaac cagtacaacg gcaaggccga gctgccgcac tcggtgttcc
3001 acggccacaa gcagctggcc accaagatcc gcctgtggca ccagcagggc gagcgctgcc
3061 tgtacaccgg caagaccatc tcgatccacg acctgatcaa caactcgaac cagttcgagg
3121 tggaccacat cctgccgctg tcgatcacct tcgacgactc gctggccaac aaggtgctgg
3181 tgtacgccac cgccaaccag gagaagggcc agcgcacccc gtaccaggcc ctggactcga
3241 tggacgacgc ctggtcgttc cgcgagctga aggccttcgt gcgcgagtcg aagaccctgt
3301 cgaacaagaa gaaggagtac ctgctgaccg aggaggacat ctcgaagttc gacgtgcgca
3361 agaagttcat cgagcgcaac ctggtggaca cccgctacgc ctcgcgcgtg gtgctgaacg
3421 ccctgcagga gcacttccgc gcccacaaga tcgacaccaa ggtgtcggtg gtgcgcggcc
3481 agttcacctc gcagctgcgc cgccactggg gcatcgagaa gacccgcgac acctaccacc
3541 accacgccgt ggacgccctg atcatcgccg cctcgtcgca gctgaacctg tggaagaagc
3601 agaagaacac cctggtgtcg tactcggagg accagctgct ggacatcgag accggcgagc
3661 tgatctcgga cgacgagtac aaggagtcgg tgttcaaggc cccgtaccag cacttcgtgg
3721 acaccctgaa gtcgaaggag ttcgaggact cgatcctgtt ctcgtaccag gtggactcga
3781 agttcaaccg caagatctcg gacgccacca tctacgccac ccgccaggcc aaggtgggca
3841 aggacaaggc cgacgagacc tacgtgctgg gcaagatcaa ggacatctac acccaggacg
3901 gctacgacgc cttcatgaag atctacaaga aggacaagtc gaagttcctg atgtaccgcc
3961 acgacccgca gaccttcgag aaggtgatcg agccgatcct ggagaactac ccgaacaagc
4021 agatcaacga caagggcaag gaggtgccgt gcaacccgtt cctgaagtac aaggaggagc
4081 acggctacat ccgcaagtac tcgaagaagg gcaacggccc ggagatcaag tcgctgaagt
4141 actacgactc gaagctgggc aaccacatcg acatcacccc gaaggactcg aacaacaagg
4201 tggtgctgca gtcggtgtcg ccgtggcgcg ccgacgtgta cttcaacaag accaccggca
4261 agtacgagat cctgggcctg aagtacgccg acctgcagtt cgacaagggc accggcacct
4321 acaagatctc gcaggagaag tacaacgaca tcaagaagaa ggagggcgtg gactcggact
4381 cggagttcaa gttcaccctg tacaagaacg acctgctgct ggtgaaggac accgagacca
4441 aggagcagca gctgttccgc ttcctgtcgc gcaccatgcc gaagcagaag cactacgtgg
4501 agctgaagcc gtacgacaag cagaagttcg agggcggcga ggccctgatc aaggtgctgg
4561 gcaacgtggc caactcgggc cagtgcaaga agggcctggg caagtcgaac atctcgatct
4621 acaaggtgcg caccgacgtg ctgggcaacc agcacatcat caagaacgag ggcgacaagc
4681 cgaagctgga cttctaaata ctctggccat ttttctttac cgcctttgag tgagctgata
4741 ccgctcgccg cagccgaacg accgagcgca acgcgtgcgg ccgctgatta gctaagcaga
4801 aggccatcct gacggatggc ctttttctag aaaaaaaaaa agcgccgcaa ctgcggcgct
4861 tttttttttt taattaaaga aatattggat cgtcggcacc gtcacggccg tgggaggcgg
4921 cacgatccgc gacgtgatga tcggccgcat ccccacggtg ctgcgcagtg agctctacgc
4981 catcccggcg ttgatctgtg cgttcgcacg cacaggcccg gtgtgagaag ggtctctgac
5041 gagcgggaga acccacccgg ggtgggcgag tttgtcctgc gtgtgctcgg tcgagtaggc
5101 tctgggagta cccgtgtgta cgaccagcac ggcatacatc atttcgacgc cgagagattc
5161 gccgcccgaa atgagcacga tccgcatggt accatcagga ggaatcactt cgcaatgtct
5221 agattagata aaagtaaagt gattaacagc gcattagagc tgcttaatga ggtcggaatc
5281 gaaggtttaa caacccgtaa actcgcccag aagcttggtg tagagcagcc tacattgtat
5341 tggcatgtaa aaaataagcg ggccctgctc gacgccttag ccattgagat gttagatagg
5401 caccatactc acttttgccc tttagaaggg gaaagctggc aagatttttt acgtaataac
5461 gctaaaagtt ttagatgtgc tttactaagt catcgcgatg gagcaaaagt acatttaggt
5521 acacggccta cagaaaaaca gtatgaaact ctcgaaaatc aattagcctt tttatgccaa
5581 caaggttttt cactagagaa cgcgttatat gcactcagcg ctgtggggca ttttacctta
5641 ggttgcgtat tggaagatca agagcatcaa gtcgctaaag aagaaaggga aacacctact
5701 actgatagta tgccgccatt attacgacaa gctatcgaat tatttgatca ccaaggtgca
5761 gagccagcct tcttattcgg ccttgaattg atcatctgcg gattagaaaa acaacttaaa
5821 tgtgaaagtg ggtcttaatg accatgggct agcgctttag ctaattaatt taaaataaaa
5881 aaggggacct ctagggtccc caattaaata gttaacacta gtattaaaaa aaaaagcccc
5941 gcgattgcgg ggcttttttt tttcgacggc gagggttccg accgctgcaa ctcccggtgc
6001 aaccttgtcc cggtctattc tcttcactgc accagctcca atctggtgtg aatgcccctc
6061 gtctgttcgc gcaggcgggg ggctctattc gtttgtcagc atcgaaagta gccagatcag
6121 ggatgcgttg caaccgcgta tgcccaggtc agaagagtcg cacaagagtt gcagacccct
6181 ggaaagaaaa atggccagag ggcgaaaaca ccctctgacc agcggagcgg gcgacgggaa
6241 tcgaacccgc gtagctagtt tggaagaatg ggtgtctgcc gaccacatat atgggccggt
6301 caagataggt ttttaccccc tctcggctgc atcctctaag tggaaagaaa ttgcaggtcg
6361 tagaagcgcg ttgaagcctg agagttgcac aggagttgca acccggtagc cttgttcacg
6421 acgagaggag acctagttgg cacgtcgcgg atggggatcg ctgaagactc agcgcagcgg
6481 gaggatccaa gcctcatacg tcaacccgca ggacggtgtg aggtactacg cgctgcagac
6541 ctacgacaac aagatggacg ccgaagcctg gctcgcgggc gagaagcggc tcatcgagat
6601 ggagacctgg acccctccac aggaccgggc gaagaaggca gccgccagcg ccatcacgct
6661 ggaggagtac acccggaagt ggctcgtgga gcgcgacctc gcagacggca ccagggatct
6721 gtacagcggg cacgcggagc gccgcatcta cccggtgcta ggtgaagtgg cggtcacaga
6781 gatgacgcca gctctggtgc gtgcgtggtg ggccgggatg ggtaggaagc acccgactgc
6841 ccgccggcat gcctacaacg tcctccgggc ggtgatgaac acagcggtcg aggacaagct
6901 gatcgcagag aacccgtgcc ggatcgagca gaaggcagcc gatgagcgcg acgtagaggc
6961 gctgacgcct gaggagctgg acatcgtcgc cgctgagatc ttcgtgcact accggatcgc
7021 ggcatacatc ctggcgtgga cgagcctccg gttcggagag ctgatcgagc ttcgccgcaa
7081 ggacatcgtg gacgacggca tgacgatgaa gctccgggtg cgccgtggcg cttcccgcgt
7141 ggggaacaag atcgtcgttg gcaacgccaa gaccgtccgg tcgaagcgtc ctgtgacggt
7201 tccgcctcac gtcgcggaga tgatccgagc gcacatgaag gaccgtacga agatgaacaa
7261 gggccccgag gcattcctgg tgaccacgac gcagggcaac cggctgtcga agtccgcgtt
7321 caccaagtcg ctgaagcgtg gctacgccaa gatcggtcgg ccggaactcc gcatccacga
7381 cctccgcgct gtcggcgcta cgttcgccgc tcaggcaggt gcgacgacca aggagctgat
7441 ggcccgcctc ggtcacacga ctcctaggat ggcgatgaag taccagatgg cgtctgaggc
7501 ccgcgacgag gctatcgctg aggcgatgtc caagctggcc aagacctcct gaaacgcaaa
7561 aagcccccct cccaaggaca ctgagtccta aagagggggg tttcttgtca gtacgcgaag
7621 aaccacgcct ggccgcgagc gccagcaccg ccgctctgtg cggagacctg ggcaccagcc
7681 ccgccgccgc caggagcatt gccgttcccg ccagctagcc acctgacgtc gggggggggg
7741 gaaagccacg ttgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
7801 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
7861 attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta
7921 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg
7981 tatgggaagc cccatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
8041 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
8101 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
8161 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
8221 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
8281 gatcgcgtat ttcgcctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
8341 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
8401 aatcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
8461 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
8521 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
8581 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
8641 catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag
8701 agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact tttgctgagt
8761 tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt
8821 tcaaaatcac caactggtcc acctacaaca aagctctcac caaccgtggc tccctcacga
8881 t
//
