LOCUS       sequence-291745-        5936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable NOT gate without RiboJ insulator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5936)
  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 5936)
  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: pVB004
FEATURES             Location/Qualifiers
     source          1..5936
                     /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        2984..3033
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(3083..3085,3086..3088,3089..3802)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3128..3149)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3389..3408)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3736..3757
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        3865..3899
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     promoter        3865..3899
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     promoter        3865..3899
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     CDS             3953..5035
                     /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(3972..3991)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5054..5677
                     /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(5127..5146)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5860..5879)
                     /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 gcacgggcgt
     2161 taatctctgg cttgctttat gtctgtaaac agaggagata cagaatgaaa gcaagcaacc
     2221 tggcggcagc gcaaaagatg cgtaaagcac gtaccccgag ccgtagcagc attggtagcc
     2281 tgcgtagtcc gcatacccat aaagcaattc tgaccagcac cattgaaatc ctgaaagaat
     2341 gtggttatag cggtctgagc attgaaagcg ttgcacgtcg tgccggtgca agcaaaccga
     2401 ccatttatcg ttggtggacc aataaagcag cactgattgc cgaagtgtat gaaaatgaaa
     2461 gcgaacaggt gcgtaaattt ccggatctgg gtagctttaa agccgatctg gattttctgc
     2521 tgcgtaatct gtggaaagtt tggcgtgaaa ccatttgtgg tgaagcattt cgttgtgtta
     2581 ttgcagaagc acagctggac cctgcaaccc tgacccagct gaaagatcag tttatggaac
     2641 gtcgtcgtga gatgccgaaa aaactggttg aaaatgccat tagcaatggt gaactgccga
     2701 aagataccaa tcgtgaactg ctgctggata tgatttttgg tttttgttgg tatcgcctgc
     2761 tgaccgaaca gctgaccgtt gaacaggata ttgaagaatt taccttcctg ctgattaatg
     2821 gtgtttgtcc gggtacacag cgttaaggaa acacagaaaa aagcccgcac ctgacagtgc
     2881 gggctttttt tttcgaccaa aggtgtcaac gttcgacgta cggtggaatc tgattcgtta
     2941 ccaattgaca tgatacgaaa cgtaccgtat cgttaaggtc aagctgtcac cggatgtgct
     3001 ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt gtttaatact
     3061 agagaaagag gggaaatact agatggtgag caagggcgag gagctgttca ccggggtggt
     3121 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     3181 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccacaggcaa
     3241 gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcctgc aatgcttcgc
     3301 ccgctacccc gaccacatga agctgcacga cttcttcaag tccgccatgc ccgaaggcta
     3361 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3421 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3481 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     3541 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     3601 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac accccaatcg gcgacggccc
     3661 cgtgctgctg cccgacaacc actaccttag ctaccagtcc gccctgagca aagaccccaa
     3721 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3781 catggacgag ctgtacaagt aactcggtac caaattccag aaaagaggcc tcccgaaagg
     3841 ggggcctttt tcgttttggt ccagtttacg gctagctcag tcctaggtat tatgctagcc
     3901 tgaagctgtc accggatgta cgccggaaga gagtcaattc agggtggtga atatgaaacc
     3961 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4021 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4081 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4141 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     4201 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     4261 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4321 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     4381 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     4441 tgaggacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     4501 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     4561 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     4621 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     4681 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     4741 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagatagct catgttatat
     4801 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     4861 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc cagtctcact
     4921 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     4981 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgataatc
     5041 caggaggaaa aaaatgtcca gattagataa aagtaaagtg attaacagcg cattagagct
     5101 gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt
     5161 agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc
     5221 cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca
     5281 agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg
     5341 agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca
     5401 attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc
     5461 tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga
     5521 agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt
     5581 atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg
     5641 attagaaaaa caacttaaat gtgaaagtgg gtcctaataa ttggtaacga atcagacaat
     5701 tgacggctcg agggagtagc atagggtttg cagaatccct gcttcgtcca tttgacaggc
     5761 acattatgca tcgatgataa gctgtcaaac atgagcagat cctctacgcc ggacgcatcg
     5821 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     5881 atggggaaga tcgggctcgc cacttcgggc tcatgagcaa atattttatc tgaggt
//
