LOCUS       sequence-149379-        4305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid - BsmBI entry sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4305)
  AUTHORS   Nadler DC, Morgan SA, Flamholz A, Kortright KE, Savage DF
  TITLE     Rapid construction of metabolite biosensors using domain-insertion 
            profiling.
  JOURNAL   Nat Commun. 2016 Jul 29;7:12266. doi: 10.1038/ncomms12266.
  PUBMED    27470466
REFERENCE   2  (bases 1 to 4305)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2016-07-29"; volume: "7:12266. doi"; pages: " 
            10.1038/ncomms12266"
COMMENT     Sequence Label: pTKEI-Dest
FEATURES             Location/Qualifiers
     source          1..4305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(379..1461)
                     /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     1423..1442
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1462..1539)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(1584..1589,1590..1606,1607..1613)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1621..1637
                     /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)."
     protein_bind    1733..1754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1769..1774,1775..1792,1793..1799)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1807..1823
                     /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)."
     primer_bind     1831..1847
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1831..1847
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1843..2166
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    1860..1916
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1917..1934)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1917..1933)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1926..1948)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2142..2161)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             2264..2281
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2299..2316)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2469..2555
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2647..2674
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(2780..3595)
                     /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(2780..3595)
                     /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     3503..3522
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3587..3606
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     4206..4225
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgccacg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
      241 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
      301 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
      361 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      421 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
      481 ttttcttttc accagtgaca cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
      541 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
      601 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
      661 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
      721 atcgttggca accagcatcg cagtgggaac gataccctca ttcagcattt gcatggtttg
      781 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
      841 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
      901 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
      961 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     1021 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     1081 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     1141 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     1201 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     1261 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     1321 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     1381 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     1441 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     1501 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     1561 tatgcgactc cgatcccgca aatttgacaa ttaatcatcc ggctcgtata atgtgtggaa
     1621 ttgtgagcgg ataacaattt cacacaagga gatataccat gtccagagac gattaatgca
     1681 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     1741 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     1801 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     1861 agcttgcatg cctgcaggtc gactctagag gatccccggg taccgagctc gaattcactg
     1921 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1981 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     2041 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     2101 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     2161 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     2221 ctgctcccgg catccgctta cagacaagct cgtctctagc ggtcaccatc accatcacca
     2281 ttaagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     2341 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     2401 agtgaaacgc cgtagcgccg atggtagtgt ggggtatccc catgcgagag tagggaactg
     2461 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     2521 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     2581 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     2641 ttaagcagaa ggccatcctg acggatggcc ttttctgaaa ggaggaacta tatccggata
     2701 tccacaggac gggtgtggtc gccatgatcg cgtagtcaag gtttcttaga cgtcaggtgg
     2761 cacttttcgg ggaaatgtgt tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2821 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2881 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2941 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     3001 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     3061 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     3121 cgttattcat tcgtgattgc gcctgagcga ggcgaaatac gcgatcactg ttaaaaggac
     3181 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     3241 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttgccg gggatcgcag
     3301 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     3361 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     3421 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     3481 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     3541 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     3601 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     3661 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcaagg
     3721 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3781 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3841 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3901 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3961 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4021 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4081 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4141 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4201 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4261 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaa
//
