LOCUS       sequence-307707-        5782 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses MphR protein under the control of T7-lacO promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5782)
  AUTHORS   Jung JK, Alam KK, Verosloff MS, Capdevila DA, Desmau M, Clauer PR, 
            Lee JW, Nguyen PQ, Pasten PA, Matiasek SJ, Gaillard JF, Giedroc DP, 
            Collins JJ, Lucks JB
  TITLE     Cell-free biosensors for rapid detection of water contaminants.
  JOURNAL   Nat Biotechnol. 2020 Jul 6. pii: 10.1038/s41587-020-0571-7. doi: 
            10.1038/s41587-020-0571-7.
  PUBMED    32632301
REFERENCE   2  (bases 1 to 5782)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Jul 6. pii: 10.1038/s41587-020-0571-7. doi: 
            10.1038/s41587-020-0571-7."
COMMENT     Sequence Label: pJBL715
FEATURES             Location/Qualifiers
     source          1..5782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     825..844
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(864..941)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1147..1166
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1250..1269
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1250..1268
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1269..1293
                     /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)."
     RBS             1308..1330
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1940..1957
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1966..1984)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1980..2027
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2064..2519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2151..2170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2361..2382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2611..3426)
                     /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             complement(2611..3426)
                     /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     3334..3353
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3418..3437
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3548..4136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4037..4056
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4290..4307
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4322..4461
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4387..4406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4547..4569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4563..4754)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5563..5782,1..863))
                     /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"
ORIGIN
        1 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
       61 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
      121 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
      181 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
      241 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
      301 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
      361 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
      421 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
      481 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
      541 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
      601 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
      661 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
      721 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
      781 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
      841 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
      901 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
      961 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     1021 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     1081 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     1141 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     1201 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     1261 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     1321 aagaaggaga tataccatgc cgcgtccgaa attaaaatcg gatgacgaag ttcttgaggc
     1381 agcgactgta gtattgaaac gctgtggtcc cattgagttt acgctttctg gggtcgcgaa
     1441 agaagttggt cttagccgcg cagcgttgat ccaacgtttt accaaccgcg acacgctgtt
     1501 ggtgcgtatg atggaacgtg gcgtggagca ggtgcgtcac tacttaaatg cgatccccat
     1561 tggggcgggc cctcagggtt tatgggagtt tcttcaagtt ttagtgcgta gcatgaatac
     1621 gcgtaatgac ttctctgtaa actacttgat ttcgtggtac gagcttcaag tccccgaatt
     1681 gcgcacactg gcgattcaac gcaaccgcgc agtagtagaa ggtatccgca agcgtttgcc
     1741 tcctggggcc cctgcggcgg ctgagttgct gctgcatagc gtgattgcgg gtgccacgat
     1801 gcagtgggcg gtggacccgg acggtgaatt agccgatcat gtattagccc agatcgctgc
     1861 gatcttgtgt ttaatgttcc ccgaacatga tgactttcag ttgcttcaac cccatgcgaa
     1921 gcttgcggcc gcactcgagc accatcacca tcaccattaa ctcgaccgct gagcaataac
     1981 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2041 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2101 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2161 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2221 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2281 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2341 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2401 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2461 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2521 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2581 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     2641 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     2701 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     2761 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     2821 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     2881 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     2941 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     3001 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     3061 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     3121 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     3181 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     3241 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     3301 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     3361 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     3421 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     3481 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3541 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3601 cactaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3661 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3721 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3781 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3841 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3901 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3961 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4021 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4081 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4141 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4201 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4261 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4321 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     4381 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4441 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4501 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4561 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     4621 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     4681 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     4741 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     4801 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4861 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4921 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4981 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     5041 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     5101 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     5161 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     5221 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     5281 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     5341 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     5401 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     5461 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     5521 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     5581 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5641 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     5701 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     5761 caggcgaaaa tcctgtttga tg
//
