LOCUS       sequence-450323-        6071 bp    DNA     circular SYN 15-APR-2025
DEFINITION  To express VapBC35(Rv1962) Toxin antitoxin in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6071)
  AUTHORS   Panagoda NT, Balazsi G, Sampson NS
  TITLE     Mycobacterium tuberculosis Mce3R TetR-like Repressor Forms an 
            Asymmetric Four-Helix Bundle and Binds a Nonpalindrome 
            Sequencedagger.
  JOURNAL   ACS Chem Biol. 2024 Dec 20;19(12):2580-2592. doi: 
            10.1021/acschembio.4c00687. Epub 2024 Nov 15.
  PUBMED    39545866
REFERENCE   2  (bases 1 to 6071)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acschembio.4c00687"; journalName: "ACS Chem Biol"; date: 
            "2024-12-20- 20"; volume: "19"; issue: "12"; pages: "2580-2592"
COMMENT     Sequence Label: pET28b-VapBC35
FEATURES             Location/Qualifiers
     source          1..6071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(105..152)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     148..166
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(219..236)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    558..582
                     /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)."
     promoter        complement(583..601)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1091..1113
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1128..1152
                     /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)."
     promoter        complement(1153..1171)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1255..1274)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1480..1557
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1558..2640
                     /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(1577..1596)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3449..3640
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3634..3656)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3742..3881
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3797..3816
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3896..3913)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4067..4655)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4147..4166)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4766..4785)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4777..5592
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4777..5592
                     /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
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4850..4869)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5684..6071,1..68))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5821..5842)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6033..6052
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
       61 gggcgcgtcc cattcgccaa tccggatata gttcctcctt tcagcaaaaa acccctcaag
      121 acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc agcagccaac
      181 tcagcttcct ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg gtggtgctcg
      241 aggtaccgct cctcggcgcg catgcgctcc aacagcgttc ccgagtcgag accgccgcgc
      301 atcggaattg tgggccgagg cgccgggcca tgcactctcg ccggttgcac actgccggtg
      361 ctgatcagtg agtcgagagg gccggcagaa gccgggatta ttcgggcgat aaccttgccg
      421 cgctcagtca ggttgatctc ttcaccgcgc ttgacgcggg ccaggacctt ggacgtctcc
      481 tggttgagcg ttcgtatgga cacctcattc accatatgta tatctccttc ttatacttaa
      541 ctaatatact aagatgggga attgttatcc gctcacaatt cccctatagt gagtcgtatt
      601 aatttcgatt atgcggccgt gtacaatacg attactttct gttcgactta agcattatgc
      661 ggccgcaagc tttcatcgcg cttgtccggg tgcgacgacc gccaacgaca gggcctcggc
      721 ggcttcctta aggcggttgt cgtaggtaac cagcgcggtc aatggtgcaa cggatccggc
      781 ggtttgagca gtggctaggt gtatcgcgtc gagcgagcgc agtgctgggt tggggtaggc
      841 cgccgcggtg gagcgtatga ccgcgtcgat ttcgaaacgg tccagcctgg ctagcacgga
      901 gggcaccgcc ggtagccctt ctggggagac tgcgcggatg gctctggata gctcaacttc
      961 ggtcaaagcc gatgtgatcc accgtagttc ggtgcggtca tcgagccaat cagctaaagc
     1021 gtcagattcg acctcgatcc gaattagctt gaccagcgcc gaggtttcca ggtagatcac
     1081 ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     1141 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct
     1201 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
     1261 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
     1321 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
     1381 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     1441 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
     1501 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     1561 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     1621 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     1681 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     1741 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     1801 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     1861 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     1921 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     1981 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     2041 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     2101 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     2161 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     2221 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     2281 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     2341 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     2401 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     2461 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     2521 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     2581 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     2641 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc
     2701 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     2761 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg
     2821 ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
     2881 ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg
     2941 tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt
     3001 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     3061 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     3121 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     3181 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     3241 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     3301 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     3361 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     3421 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     3481 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     3541 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     3601 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3661 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3721 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3781 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg
     3841 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     3901 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3961 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4021 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4081 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4141 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4201 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4261 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4321 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4381 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4441 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4501 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4561 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4621 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4681 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa
     4741 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     4801 tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     4861 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     4921 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     4981 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     5041 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag
     5101 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     5161 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     5221 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     5281 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     5341 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     5401 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     5461 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     5521 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     5581 gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5641 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     5701 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5761 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5821 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5881 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5941 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     6001 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     6061 agggcgctgg c
//
