LOCUS       sequence-248940-        6142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TES with RiboJ insulator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6142)
  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 6142)
  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: pVB001
FEATURES             Location/Qualifiers
     source          1..6142
                     /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"
     misc_RNA        2157..2206
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(2322..2324,2325..3038)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2364..2385)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2625..2644)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2972..2993
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        3120..3154
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     RBS             3163..3174
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4030..4041
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4159..5241
                     /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(4178..4197)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5260..5883
                     /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(5333..5352)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(6066..6085)
                     /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 gcacagctgt
     2161 caccggatgt gctttccggt ctgatgagtc cgtgaggacg aaacagcctc tacaaataat
     2221 tttgtttaag ggcgttaatc tctggcttgc tttatgtctg taaacagagg agatacagaa
     2281 tgaaagcaag caacctggcg gcagcgcaaa agatgcgtaa agtgagcaag ggcgaggagc
     2341 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2401 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2461 tctgcaccac aggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg
     2521 gcctgcaatg cttcgcccgc taccccgacc acatgaagct gcacgacttc ttcaagtccg
     2581 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2641 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2701 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2761 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2821 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2881 caatcggcga cggccccgtg ctgctgcccg acaaccacta ccttagctac cagtccgccc
     2941 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     3001 ccgggatcac tctcggcatg gacgagctgt acaagtaact cggtaccaaa ttccagaaaa
     3061 gaggcctccc gaaagggggg ccttttttcg ttttggtcca atggtcacca tatatcaagt
     3121 ttacggctag ctcagtccta ggtactatgc tagctactag agaaagagga gaaatactag
     3181 atggctgaag cgcaaaatga tcccctgctg ccgggatact cgtttaatgc ccatctggtg
     3241 gcgggtttaa cgccgattga ggccaacggt tatctcgatt tttttatcga ccgaccgctg
     3301 ggaatgaaag gttatattct caatctcacc attcgcggtc agggggtggt gaaaaatcag
     3361 ggacgagaat ttgtttgccg accgggtgat attttgctgt tcccgccagg agagattcat
     3421 cactacggtc gtcatccgga ggctcgcgaa tggtatcacc agtgggttta ctttcgtccg
     3481 cgcgcctact ggcatgaatg gcttaactgg ccgtcaatat ttgccaatac ggggttcttt
     3541 cgcccggatg aagcgcacca gccgcatttc agcgacctgt ttgggcaaat cattaacgcc
     3601 gggcaagggg aagggcgcta ttcggagctg ctggcgataa atctgcttga gcaattgtta
     3661 ctgcggcgca tggaagcgat taacgagtcg ctccatccac cgatggataa tcgggtacgc
     3721 gaggcttgtc agtacatcag cgatcacctg gcagacagca attttgatat cgccagcgtc
     3781 gcacagcatg tttgcttgtc gccgtcgcgt ctgtcacatc ttttccgcca gcagttaggg
     3841 attagcgtct taagctggcg cgaggaccaa cgtatcagcc aggcgaagct gcttttgagc
     3901 accacccgga tgcctatcgc caccgtcggt cgcaatgttg gttttgacga tcaactctat
     3961 ttctcgcggg tatttaaaaa atgcaccggg gccagcccga gcgagttccg tgccggttaa
     4021 taaccaatta ttgaaggccg ctaacgcagc ctttttttgt ttctggtctc ccaatggcgg
     4081 cgcgccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     4141 caattcaggg tggtgaatat gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     4201 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     4261 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     4321 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     4381 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     4441 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     4501 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     4561 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     4621 cccatcaaca gtattatttt ctcccatgag gacggtacgc gactgggcgt ggagcatctg
     4681 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     4741 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     4801 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     4861 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     4921 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     4981 gataccgaag atagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     5041 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     5101 ggcaatcagc tgttgccagt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     5161 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     5221 cgactggaaa gcgggcagtg acaatccagg aggaaaaaaa tgtccagatt agataaaagt
     5281 aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc
     5341 cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat
     5401 aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt
     5461 tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga
     5521 tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa
     5581 aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta
     5641 gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa
     5701 gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg
     5761 ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta
     5821 ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtcc
     5881 taataattgg taacgaatca gacaattgac ggctcgaggg agtagcatag ggtttgcaga
     5941 atccctgctt cgtccatttg acaggcacat tatgcatcga tgataagctg tcaaacatga
     6001 gcagatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
     6061 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat
     6121 gagcaaatat tttatctgag gt
//
