LOCUS       sequence-248936-        6008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable NOT gate with RiboJ insulator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6008)
  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 6008)
  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: pVB002
FEATURES             Location/Qualifiers
     source          1..6008
                     /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
                     /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
                     SDFDDERNGWPVEQVWKEMHKLLLFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     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)"
     misc_RNA        3059..3108
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(3158..3160,3161..3163,3164..3877)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3203..3224)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3464..3483)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3811..3832
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4025..5107
                     /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(4044..4063)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5126..5749
                     /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(5199..5218)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5932..5951)
                     /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 tggaagatgc 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 tgagaatagc
     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 agcacgtacc ccgagccgta
     2341 gcagcattgg tagcctgcgt agtccgcata cccataaagc aattctgacc agcaccattg
     2401 aaatcctgaa agaatgtggt tatagcggtc tgagcattga aagcgttgca cgtcgtgccg
     2461 gtgcaagcaa accgaccatt tatcgttggt ggaccaataa agcagcactg attgccgaag
     2521 tgtatgaaaa tgaaagcgaa caggtgcgta aatttccgga tctgggtagc tttaaagccg
     2581 atctggattt tctgctgcgt aatctgtgga aagtttggcg tgaaaccatt tgtggtgaag
     2641 catttcgttg tgttattgca gaagcacagc tggaccctgc aaccctgacc cagctgaaag
     2701 atcagtttat ggaacgtcgt cgtgagatgc cgaaaaaact ggttgaaaat gccattagca
     2761 atggtgaact gccgaaagat accaatcgtg aactgctgct ggatatgatt tttggttttt
     2821 gttggtatcg cctgctgacc gaacagctga ccgttgaaca ggatattgaa gaatttacct
     2881 tcctgctgat taatggtgtt tgtccgggta cacagcgtta aggaaacaca gaaaaaagcc
     2941 cgcacctgac agtgcgggct ttttttttcg accaaaggtg tcaacgttcg acgtacggtg
     3001 gaatctgatt cgttaccaat tgacatgata cgaaacgtac cgtatcgtta aggtcaagct
     3061 gtcaccggat gtgctttccg gtctgatgag tccgtgagga cgaaacagcc tctacaaata
     3121 attttgttta atactagaga aagaggggaa atactagatg gtgagcaagg gcgaggagct
     3181 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     3241 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     3301 ctgcaccaca ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg
     3361 cctgcaatgc ttcgcccgct accccgacca catgaagctg cacgacttct tcaagtccgc
     3421 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     3481 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     3541 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     3601 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     3661 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     3721 aatcggcgac ggccccgtgc tgctgcccga caaccactac cttagctacc agtccgccct
     3781 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3841 cgggatcact ctcggcatgg acgagctgta caagtaactc ggtaccaaat tccagaaaag
     3901 aggcctcccg aaaggggggc cttttttcgt tttggtccaa tggcggcgcg ccatcgaatg
     3961 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     4021 gaatatgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     4081 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     4141 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     4201 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     4261 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     4321 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     4381 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     4441 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     4501 tattttctcc catgaggacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     4561 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     4621 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     4681 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     4741 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     4801 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagatag
     4861 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     4921 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     4981 gccagtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     5041 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     5101 gcagtgataa tccaggagga aaaaaatgtc cagattagat aaaagtaaag tgattaacag
     5161 cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta aactcgccca
     5221 gaagctaggt gtagagcagc ctacattgta ttggcatgta aaaaataagc gggctttgct
     5281 cgacgcctta gccattgaga tgttagatag gcaccatact cacttttgcc ctttagaagg
     5341 ggaaagctgg caagattttt tacgtaataa cgctaaaagt tttagatgtg ctttactaag
     5401 tcatcgcgat ggagcaaaag tacatttagg tacacggcct acagaaaaac agtatgaaac
     5461 tctcgaaaat caattagcct ttttatgcca acaaggtttt tcactagaga atgcattata
     5521 tgcactcagc gctgtggggc attttacttt aggttgcgta ttggaagatc aagagcatca
     5581 agtcgctaaa gaagaaaggg aaacacctac tactgatagt atgccgccat tattacgaca
     5641 agctatcgaa ttatttgatc accaaggtgc agagccagcc ttcttattcg gccttgaatt
     5701 gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtcctaat aattggtaac
     5761 gaatcagaca attgacggct cgagggagta gcatagggtt tgcagaatcc ctgcttcgtc
     5821 catttgacag gcacattatg catcgatgat aagctgtcaa acatgagcag atcctctacg
     5881 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     5941 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc aaatatttta
     6001 tctgaggt
//
