LOCUS       sequence-128548-        9083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This E. coli DH10B strain harbors a shuttle vector plasmid that 
            expresses the Mixed Feedback Loop version of the UBER system with a 
            T7RNAP translation rate of 300 and a TetR translation rate of 469.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9083)
  AUTHORS   Kushwaha M, Salis HM
  TITLE     A portable expression resource for engineering cross-species genetic
            circuits and pathways.
  JOURNAL   Nat Commun. 2015 Jul 17;6:7832. doi: 10.1038/ncomms8832.
  PUBMED    26184393
REFERENCE   2  (bases 1 to 9083)
  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: "2015-07-17"; volume: "6:7832. doi"; pages: " 
            10.1038/ncomms8832"
COMMENT     Sequence Label: MK1282
FEATURES             Location/Qualifiers
     source          1..9083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        190..410
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             411..1214
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1343..1798)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1480..1501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1692..1711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(2681..2701)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(2776..2794)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3291..3309
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3398..3418
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6087..6158
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6174..6201
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6263..6279)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6264..6280)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(6309..6327)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6348..6364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6348..6364)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6361..6383)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6372..6388
                     /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)."
     promoter        complement(join(6396..6402,6403..6420,6421..6426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(6579..6596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6750..7338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(7509..8300,8301..8369))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8132..8151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8370..8474)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    8511..9014
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc
      241 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
      301 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
      361 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag
      421 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
      481 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
      541 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
      601 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
      661 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc
      721 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg
      781 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac
      841 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat
      901 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg
      961 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg
     1021 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg
     1081 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg
     1141 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg
     1201 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta
     1261 gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc acagatgcgt
     1321 aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa attcgcgtta
     1381 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1441 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1501 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1561 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggggccg taaagcacta
     1621 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     1681 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     1741 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     1801 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     1861 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     1921 tttcccagtc acgacgttgt aaacgacggc cagtgaattg gcgaattgga gctattaatt
     1981 aaacaaaaaa cccctacccc cccgatatgt atttattgcg aaaaaacccc gccgaagcgg
     2041 ggttttttgc gaccggttat tatgaacctg attcacattt taactgtttc tccaaaccgc
     2101 aaatgattaa ttctaaacca aataaaaatg caggttctgc accttgatga tcaaataatt
     2161 caattgcttg acgtaataaa ggaggcattg aatctgttgt aggtgtttct ctttcctcct
     2221 tggcaacttg atgttcttga tcttctaaaa cacaacctaa ggtaaaatga ccaactgctg
     2281 ataatgcata taatgcgttt tctaatgaaa aaccttgttg acataagaat gctaactgat
     2341 tttctaatgt ttcatattgt ttttctgtag gacgtgtacc taagtgaact ttggcaccat
     2401 ctctatgtga taataatgca caacgaaatg atttggcatt gttacgtaag aagtcttgcc
     2461 atgattctcc ttctaatgga caaaaatgtg tatgatgtct atctaacatt tcgattgcta
     2521 atgcatctaa taatgcacgt ttgtttttaa catgccaata taatgtaggt tgttcaacac
     2581 ctaatttctg tgctaattta cgtgtagtta aaccttcgat accaacttca tttaataatt
     2641 ctaatgctga gttaataact tttgatttat ctaaacgtga aactttacgt ttttttttag
     2701 gaggatagat ctccatggtg gtagacattt ttatgtgcct ctaactcttg ctattaatag
     2761 gcgccccggg atagccctat agtgagtcgt attaatttcc gcgggcgagc tcccattatt
     2821 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattg
     2881 atctgtcgac aggaactgga cagcgtgtcg aaaaagctcg acaggaactg gacagcgtgt
     2941 cgaaaaagct cgacaggaac tggacagcgt gtcgaaaaag ctcgacagga actggacagc
     3001 gtgtcgaaaa agctcgagca gatccgccag gcgtgtatat atagcgtgga tggccaggca
     3061 actttagtgc tgacacatac aggcatatat atatgtgtgc gacgacacat gatcatatgg
     3121 catgcatgtg ctctgtatgt atataaaact cttgttttct tcttttctct aaatattctt
     3181 tccttataca ttaggtcctt tgtagcataa attactatac ttctatagac acgcaaacac
     3241 aaatacacac actaaattaa taactagttc tatcattgat aggtataaat taatacgact
     3301 cactataggg agacctatca gtgatagagg atccaaccga gcccaatata ggactcaggg
     3361 tgccaaaaaa atgtctacca ccatgggaat tcaacctcct aaaaagaaac gtaaagttaa
     3421 tactattaat attgctaaaa atgacttctc agatattgaa ttagcagcca ttccatttaa
     3481 tacattagca gatcactatg gtgaacgttt agcacgtgaa cagttagcat tagaacatga
     3541 atcatatgaa atgggtgaag cacgttttcg taagatgttc gagcgtcagt taaaagcagg
     3601 tgaagttgca gataatgcag cagccaaacc tttaattact acattattac ctaaaatgat
     3661 tgctcgtatt aacgattggt ttgaagaggt taaagcaaag cgtggtaaac gtcctacagc
     3721 atttcagttc ttacaagaaa tcaaacctga agcagttgca tatattacta ttaaaacaac
     3781 attagcatgt ttaacatcag cagataatac aacagttcaa gcagttgcat cagcaattgg
     3841 tcgtgcaatt gaagatgaag cacgttttgg tcgtattcgt gatttagaag ccaaacattt
     3901 taaaaaaaat gttgaagaac agttaaacaa acgtgttggt catgtttata aaaaagcatt
     3961 tatgcaggtt gttgaagcag atatgttatc aaaaggttta ttaggtggtg aagcatggtc
     4021 atcatggcat aaagaagatt caattcatgt tggtgttcgt tgtattgaaa tgttaattga
     4081 atcaacaggt atggtttcat tacatcgtca gaatgcaggt gttgttggtc aagattcaga
     4141 aacaattgaa ttagcacctg aatatgcaga agcaattgca acacgtgcag gtgcattagc
     4201 aggtatttca ccaatgtttc agccttgtgt tgttcctcct aaaccttgga caggtattac
     4261 aggtggtggt tattgggcaa atggtcgtcg tcctttagca ttagttcgta cacattcaaa
     4321 aaaagcatta atgcgttatg aagatgttta catgcctgaa gtttataaag ccattaatat
     4381 tgcacagaat actgcatgga aaatcaacaa gaaagtttta gcagttgcaa atgttattac
     4441 taaatggaaa cattgtcctg ttgaagatat tcctgcaatt gaacgtgaag aattaccaat
     4501 gaaacctgaa gatattgata tgaatcctga agcattaaca gcatggaaac gtgcagcagc
     4561 agccgtttat cgtaaagata aagcacgtaa atcacgtcgt atttcattag agttcatgtt
     4621 agagcaagca aacaagttcg caaatcataa agccatttgg tttccttata atatggattg
     4681 gcgtggtcgt gtttatgcag tttcaatgtt taatcctcaa ggtaatgata tgaccaaagg
     4741 tttattaacc ttagctaaag gtaaacctat tggtaaagaa ggttattatt ggttaaaaat
     4801 ccatggtgca aattgtgcag gtgttgataa agttcctttt ccagaacgta ttaagttcat
     4861 tgaagaaaat catgaaaata ttatggcatg tgctaaatca ccattagaaa atacatggtg
     4921 ggcagaacaa gattcacctt tttgtttttt agccttttgt tttgaatatg caggtgttca
     4981 acatcatggt ttatcatata attgttcatt accattagca tttgatggtt catgttcagg
     5041 tattcagcat ttttcagcaa tgttacgtga tgaagttggt ggtcgtgcag ttaacttatt
     5101 accttcagaa acagttcaag atatctatgg tattgttgct aaaaaagtta atgaaatctt
     5161 acaggcagat gccattaatg gtacagataa tgaagttgtt acagttacag atgaaaatac
     5221 aggtgaaata tcagaaaaag ttaaattagg taccaaagca ttagcaggtc aatggttagc
     5281 atatggtgtt acacgttcag ttactaagcg ttcagttatg acattagcat atggttcaaa
     5341 agagttcggt tttcgtcaac aggttttaga agataccatt caacctgcaa ttgattcagg
     5401 taaaggttta atgtttactc aacctaatca agcagcaggt tatatggcca aattaatatg
     5461 ggaatcagtt tcagttacag ttgttgcagc agttgaagca atgaattggt taaaatcagc
     5521 agccaaatta ttagcagcag aagttaaaga taaaaaaaca ggtgaaatct tacgtaagcg
     5581 ttgtgcagtt cattgggtta cacctgatgg ttttcctgtt tggcaagaat ataaaaaacc
     5641 tattcaaaca cgtttaaact taatgttttt aggtcagttc cgtttacaac ctactattaa
     5701 tacaaacaaa gattcagaaa ttgatgcaca taaacaagaa tcaggtattg cacctaactt
     5761 cgttcattca caagatggtt cacatttacg taaaacagtt gtttgggcac atgaaaaata
     5821 tggtattgaa tcatttgcat taattcacga ttcatttggt accattccag cagatgcagc
     5881 aaacttattc aaagcagttc gtgaaacaat ggttgataca tatgaatcat gtgatgtttt
     5941 agcagacttc tatgatcagt tcgcagatca gttacatgaa tcacagttag ataaaatgcc
     6001 tgcattacct gccaaaggta acttaaactt acgtgatatt ttagaatcag acttcgcatt
     6061 tgcctaataa ggcgcgcccc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     6121 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac
     6181 cttcgggtgg gcctttctgc gtttatagct attactcgaa cccctagccc gctcttatcg
     6241 ggcggctagg ggttttttgt atcgataccg tcgacctcga gggggggccc ggtacccagc
     6301 ttttgttccc tttagtgagg gttaattccg agcttggcgt aatcatggtc atagctgttt
     6361 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca taggagccgg aagcataaag
     6421 tgtaaagcct ggggtgccta atgagtgagg taactcacat taattgcgtt gcgctcactg
     6481 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6541 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6601 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6661 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6721 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tcggcccccc tgacgagcat
     6781 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6841 gcgttccccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6901 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     6961 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     7021 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     7081 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     7141 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     7201 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     7261 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     7321 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     7381 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     7441 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     7501 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     7561 tcatccatag ttgcctgact gcccgtcgtg tagataacta cgatacggga gggcttacca
     7621 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7681 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7741 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7801 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7861 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtga
     7921 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7981 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     8041 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     8101 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     8161 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     8221 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     8281 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     8341 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     8401 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     8461 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ggtccttttc atcacgtgct
     8521 ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa atagaaagta
     8581 aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag actctagggg
     8641 gatcgccaac aaatactacc ttttatcttg ctcttcctgc tctcaggtat taatgccgaa
     8701 ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat cctgtgattt tacattttac
     8761 ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata aatatatatg
     8821 taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt cttcattccg
     8881 taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat aaaaataaat
     8941 aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg cgtgtaagtt
     9001 acaggcaagc gatccgtcct aagaaaccat tattatcatg acattaacct ataaaaatag
     9061 gcgtatcacg aggccctttc gtc
//
