LOCUS       sequence-406223-        4545 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpressing 6His-TetR in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4545)
  AUTHORS   Deng Y, Beahm DR, Ran X, Riley TG, Sarpeshkar R
  TITLE     Rapid modeling of experimental molecular kinetics with simple 
            electronic circuits instead of with complex differential equations.
  JOURNAL   Front Bioeng Biotechnol. 2022 Sep 28;10:947508. doi: 
            10.3389/fbioe.2022.947508. eCollection 2022.
  PUBMED    36246369
REFERENCE   2  (bases 1 to 4545)
  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: "Front 
            Bioeng Biotechnol."; date: "2022-09-28"; volume: "10:947508. doi"; 
            pages: " 10.3389/fbioe.2022.947508. eCollection 2022"
COMMENT     Sequence Label: pDA303
FEATURES             Location/Qualifiers
     source          1..4545
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..20
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(44..58,59..64,65..81,82..88)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    64..80
                     /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    96..112
                     /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)."
     CDS             160..177
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             181..804
                     /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(254..273)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      824..918
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(966..985)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             977..1792
                     /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             977..1792
                     /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(1050..1069)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2011..2097
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2190..3272)
                     /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     3234..3253
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3273..3350)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3434..3456)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3542..3682
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3597..3616
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3697..3714)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3868..4456)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3948..3967)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaataggcgt atcacgaggc cctttcgtct tcacctcgag aaatcataaa aaatttattt
       61 gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac aatttcacac
      121 agaattcatt ttattataag gaggactttt atgagcggcc atcaccatca tcatcacgga
      181 atgtccagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc
      241 ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca
      301 ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta
      361 gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt
      421 aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat
      481 ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta
      541 tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt
      601 actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca
      661 cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa
      721 ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa
      781 cttaaatgtg aaagtgggtc ctaataagct ttagctgagc ttggactcct gttgatagat
      841 ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg
      901 ttttttattg gtgagaatcc aagctagcca tgaaaataaa ctgtctgctt acataaacag
      961 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
     1021 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     1081 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
     1141 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     1201 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcttg
     1261 gttactcacg actgcgatcc ccggcaaaac agcattccag gtattagaag aatatcctga
     1321 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     1381 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     1441 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     1501 tgaacaagtc tggaaagaaa tgcacaaact tttgccattc tcaccggatt cagtcgtcac
     1561 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     1621 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     1681 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     1741 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat
     1801 tcatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
     1861 atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
     1921 gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc ccttaaacgc
     1981 ctggggtaat gactctctag cttgaggcat caaataaaac gaaaggctca gtcgaaagac
     2041 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     2101 ccctctagat tacgtgcagt cgatgataag ctgtcaaaca tgagaattgt gcctaatgag
     2161 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2221 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggg
     2281 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     2341 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     2401 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     2461 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     2521 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     2581 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     2641 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     2701 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     2761 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     2821 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     2881 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     2941 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     3001 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     3061 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     3121 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     3181 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     3241 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     3301 gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc ggaatttcgg
     3361 gcagcgttgg gtcctggcca cgggtgcgca tgatctagag ctgcctcgcg cgtttcggtg
     3421 atgacggtga aaacctctga cacatgcagc tcccggcatc cgcttacaga caagctgtga
     3481 ccgtctccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     3541 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     3601 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     3661 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3721 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3781 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3841 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3901 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3961 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4021 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4081 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4141 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4201 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4261 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4321 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4381 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4441 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4501 cgaaaactca cgttaaggga ttttggtcat gacattaacc tataa
//
