LOCUS       sequence-334212-        6833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Unregulated ""open-loop"" CMV promoter plasmid. This plasmid encodes
            eGFP to report circuit output levels. This plasmid has additional 
            sequences in the UTRs to increase gene expression levels..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6833)
  AUTHORS   Yang J, Lee J, Land MA, Lai S, Igoshin OA, St-Pierre F
  TITLE     A synthetic circuit for buffering gene dosage variation between 
            individual mammalian cells.
  JOURNAL   Nat Commun. 2021 Jul 5;12(1):4132. doi: 10.1038/s41467-021-23889-0.
  PUBMED    34226556
REFERENCE   2  (bases 1 to 6833)
  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: "2021-07-5"; volume: "12(1):4132. doi"; pages: " 
            10.1038/s41467-021-23889-0"
COMMENT     Sequence Label: CMV+ plasmid
FEATURES             Location/Qualifiers
     source          1..6833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    814..832
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    840..858
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          1041..1613
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     regulatory      1625..1634
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1631..1633,1634..1636,1637..2350)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1631..1633,1634..1636,1637..2347)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1676..1697)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1937..1956)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2284..2305
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2412..2432)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(2785..2802)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2791..3015
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3148..3167)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3299..3346
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3354..4376
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3354..4376
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4506..4627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4543..4562)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4597..4616
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4676..4692)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4676..4692)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4689..4711)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4700..4716
                     /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(4724..4730,4731..4748,4749..4754))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4769..4790
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4907..4924)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5078..5666)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5158..5177)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5837..6628,6629..6697))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6460..6479
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6698..6802)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcatccctat cagtgataga gatcagatct
      841 ccctatcagt gatagagagc tgttttgacc tccatagaag acaccgggac cgatccagcc
      901 tccggactct agcgtttaaa cttaagctgg gtacccgggg atcctctagg gcctctgagc
      961 tattccagaa gtagtgaaga ggcttttttg gaggcctagg cttttgcaaa aagctccgga
     1021 tcgatcctga gaacttcagg gtgagtttgg ggacccttga ttgttctttc tttttcgcta
     1081 ttgtaaaatt catgttatat ggagggggca aagttttcag ggtgttgttt agaatgggaa
     1141 gatgtccctt gtatcaccat ggaccctcat gataattttg tttctttcac tttctactct
     1201 gttgacaacc attgtctcct cttattttct tttcattttc tgtaactttt tcgttaaact
     1261 ttagcttgca tttgtaacga atttttaaat tcacttttgt ttatttgtca gattgtaagt
     1321 actttctcta atcacttttt tttcaaggca atcagggtat attatattgt acttcagcac
     1381 agttttagag aacaattgtt ataattaaat gataaggtag aatatttctg catataaatt
     1441 ctggctggcg tggaaatatt cttattggta gaaacaacta catcctggtc atcatcctgc
     1501 ctttctcttt atggttacaa tgatatacac tgtttgagat gaggataaaa tactctgagt
     1561 ccaaaccggg cccctctgct aaccatgttc atgccttctt ctttttccta caggtcctgc
     1621 aggcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1681 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1741 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1801 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1861 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1921 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1981 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2041 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     2101 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     2161 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2221 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2281 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2341 gtacaagtag gcggccgcaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     2401 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     2461 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     2521 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     2581 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     2641 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     2701 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcctc gagtctagag
     2761 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2821 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2881 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2941 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3001 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     3061 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3121 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3181 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3241 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga
     3301 agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc
     3361 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     3421 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     3481 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     3541 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     3601 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     3661 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     3721 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     3781 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     3841 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     3901 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     3961 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     4021 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     4081 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     4141 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     4201 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     4261 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     4321 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg
     4381 tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4441 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4501 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4561 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4621 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4681 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4741 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4801 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4861 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4921 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4981 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5041 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5101 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5161 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5221 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5281 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5341 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5401 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5461 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5521 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5581 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5641 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5701 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5761 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5821 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5881 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5941 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6001 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6061 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6121 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6181 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6241 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6301 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6361 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6421 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6481 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6541 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6601 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6661 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6721 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6781 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
