LOCUS       sequence-334452-        5396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Unregulated ""open-loop"" PGK promoter plasmid. This plasmid encodes
            eGFP to report circuit output levels..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5396)
  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 5396)
  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: PGK plasmid
FEATURES             Location/Qualifiers
     source          1..5396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..517
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(53..76)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     450..469
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      529..538
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(535..537,538..540,541..1254)
                     /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(535..537,538..540,541..1251)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(580..601)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(841..860)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1188..1209
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1348..1365)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1354..1578
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1711..1730)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1862..1909
                     /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             1917..2939
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1917..2939
                     /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    3069..3190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3106..3125)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3160..3179
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3239..3255)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3239..3255)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3252..3274)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3263..3279
                     /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(3287..3293,3294..3311,3312..3317))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3332..3353
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3470..3487)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3641..4229)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3721..3740)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4400..5191,5192..5260))
                     /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     5023..5042
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5261..5365)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
       61 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
      121 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
      181 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
      241 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
      301 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
      361 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
      421 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
      481 cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcatcccgc caccatggtg
      541 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      601 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      661 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      721 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
      781 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      841 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      901 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      961 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1021 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1081 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1141 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1201 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtagggatcc
     1261 actagtccag tgtggtggaa ttctgcagat atccagcaca gtggcggccg ctcgagtcta
     1321 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1381 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1441 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     1501 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     1561 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     1621 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1681 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1741 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1801 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     1861 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     1921 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     1981 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     2041 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     2101 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     2161 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     2221 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     2281 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     2341 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     2401 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     2461 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     2521 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     2581 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     2641 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     2701 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     2761 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     2821 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     2881 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     2941 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3001 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3061 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3121 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3181 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     3241 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3301 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3361 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3421 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3481 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3541 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3601 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3661 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3721 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3781 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3841 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3901 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3961 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4021 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4081 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4141 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4201 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4261 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4321 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4381 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4441 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4501 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4561 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4621 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4681 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4741 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4801 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4861 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4921 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4981 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5041 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5101 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5161 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5221 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5281 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5341 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
