LOCUS       sequence-248942-        6067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TES without RiboJ insulator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6067)
  AUTHORS   Bartoli V, Meaker GA, di Bernardo M, Gorochowski TE
  TITLE     Tunable genetic devices through simultaneous control of 
            transcription and translation.
  JOURNAL   Nat Commun. 2020 Apr 29;11(1):2095. doi: 10.1038/s41467-020-15653-7.
  PUBMED    32350250
REFERENCE   2  (bases 1 to 6067)
  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: "2020-04-29"; volume: "11(1):2095. doi"; pages: " 
            10.1038/s41467-020-15653-7"
COMMENT     Sequence Label: pVB003
FEATURES             Location/Qualifiers
     source          1..6067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(308..853)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(388..407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(942..1757)
                     /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(942..1757)
                     /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     1665..1684
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1749..1768
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        join(1915..1920,1921..1936,1937..1943)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1949..1968
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     protein_bind    2101..2119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2126..2144
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(2247..2249,2250..2963)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2289..2310)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2550..2569)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2897..2918
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        3045..3079
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     RBS             3088..3099
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3955..3966
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4084..5166
                     /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(4103..4122)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5185..5808
                     /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(5258..5277)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5991..6010)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcttcctcgc tcactgactc gctgcacgag gcagacctca gcgctagcgg agtgtatact
       61 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
      121 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
      181 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
      241 ggagatttcc cggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
      301 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
      361 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
      421 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
      481 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
      541 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
      601 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
      661 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
      721 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
      781 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
      841 aaaacgatct caagaagatc atcttattaa ggggtctgac gctcagtgga acgaaaaatc
      901 aatctaaagt atatatgagt aaacttggtc tgacagttac cttagaaaaa ctcatcgagc
      961 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1021 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1081 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1141 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     1201 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     1261 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     1321 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     1381 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     1441 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     1501 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1561 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     1621 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     1681 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca agacgtttcc
     1741 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     1801 gttcatgatg atatattttt atcttgtgca atgtacatca gagattttga gacacaacca
     1861 attattgaag gcctccctaa cggggggcct ttttttgaac gatcgttggc tgtgttgaca
     1921 attaatcatc ggctcgtata atgtgtggaa ttgtgagcgc tcacaattgg gtcatgactg
     1981 ggacacgcca gtcatgagaa tacagacata aagcaagcca gagattaacg aagccaatta
     2041 ttgaacaccc ttcggggtgt ttttttgttt ctggtctacc tactccaccg ttggcttttt
     2101 tccctatcag tgatagagat tgacatccct atcagtgata gagataatga gcacgggcgt
     2161 taatctctgg cttgctttat gtctgtaaac agaggagata cagaatgaaa gcaagcaacc
     2221 tggcggcagc gcaaaagatg cgtaaagtga gcaagggcga ggagctgttc accggggtgg
     2281 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2341 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accacaggca
     2401 agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg caatgcttcg
     2461 cccgctaccc cgaccacatg aagctgcacg acttcttcaa gtccgccatg cccgaaggct
     2521 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2581 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2641 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2701 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2761 aggacggcag cgtgcagctc gccgaccact accagcagaa caccccaatc ggcgacggcc
     2821 ccgtgctgct gcccgacaac cactacctta gctaccagtc cgccctgagc aaagacccca
     2881 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2941 gcatggacga gctgtacaag taactcggta ccaaattcca gaaaagaggc ctcccgaaag
     3001 gggggccttt tttcgttttg gtccaatggt caccatatat caagtttacg gctagctcag
     3061 tcctaggtac tatgctagct actagagaaa gaggagaaat actagatggc tgaagcgcaa
     3121 aatgatcccc tgctgccggg atactcgttt aatgcccatc tggtggcggg tttaacgccg
     3181 attgaggcca acggttatct cgattttttt atcgaccgac cgctgggaat gaaaggttat
     3241 attctcaatc tcaccattcg cggtcagggg gtggtgaaaa atcagggacg agaatttgtt
     3301 tgccgaccgg gtgatatttt gctgttcccg ccaggagaga ttcatcacta cggtcgtcat
     3361 ccggaggctc gcgaatggta tcaccagtgg gtttactttc gtccgcgcgc ctactggcat
     3421 gaatggctta actggccgtc aatatttgcc aatacggggt tctttcgccc ggatgaagcg
     3481 caccagccgc atttcagcga cctgtttggg caaatcatta acgccgggca aggggaaggg
     3541 cgctattcgg agctgctggc gataaatctg cttgagcaat tgttactgcg gcgcatggaa
     3601 gcgattaacg agtcgctcca tccaccgatg gataatcggg tacgcgaggc ttgtcagtac
     3661 atcagcgatc acctggcaga cagcaatttt gatatcgcca gcgtcgcaca gcatgtttgc
     3721 ttgtcgccgt cgcgtctgtc acatcttttc cgccagcagt tagggattag cgtcttaagc
     3781 tggcgcgagg accaacgtat cagccaggcg aagctgcttt tgagcaccac ccggatgcct
     3841 atcgccaccg tcggtcgcaa tgttggtttt gacgatcaac tctatttctc gcgggtattt
     3901 aaaaaatgca ccggggccag cccgagcgag ttccgtgccg gttaataacc aattattgaa
     3961 ggccgctaac gcagcctttt tttgtttctg gtctcccaat ggcggcgcgc catcgaatgg
     4021 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     4081 aatatgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     4141 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     4201 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     4261 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     4321 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     4381 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     4441 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     4501 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     4561 attttctccc atgaggacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     4621 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     4681 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     4741 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     4801 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     4861 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagatagc
     4921 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     4981 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     5041 ccagtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     5101 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     5161 cagtgacaat ccaggaggaa aaaaatgtcc agattagata aaagtaaagt gattaacagc
     5221 gcattagagc tgcttaatga ggtcggaatc gaaggtttaa caacccgtaa actcgcccag
     5281 aagctaggtg tagagcagcc tacattgtat tggcatgtaa aaaataagcg ggctttgctc
     5341 gacgccttag ccattgagat gttagatagg caccatactc acttttgccc tttagaaggg
     5401 gaaagctggc aagatttttt acgtaataac gctaaaagtt ttagatgtgc tttactaagt
     5461 catcgcgatg gagcaaaagt acatttaggt acacggccta cagaaaaaca gtatgaaact
     5521 ctcgaaaatc aattagcctt tttatgccaa caaggttttt cactagagaa tgcattatat
     5581 gcactcagcg ctgtggggca ttttacttta ggttgcgtat tggaagatca agagcatcaa
     5641 gtcgctaaag aagaaaggga aacacctact actgatagta tgccgccatt attacgacaa
     5701 gctatcgaat tatttgatca ccaaggtgca gagccagcct tcttattcgg ccttgaattg
     5761 atcatatgcg gattagaaaa acaacttaaa tgtgaaagtg ggtcctaata attggtaacg
     5821 aatcagacaa ttgacggctc gagggagtag catagggttt gcagaatccc tgcttcgtcc
     5881 atttgacagg cacattatgc atcgatgata agctgtcaaa catgagcaga tcctctacgc
     5941 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     6001 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagca aatattttat
     6061 ctgaggt
//
