LOCUS       sequence-334456-        7259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Standalone IFF circuit plasmid that has the same implementation of 
            the IFF subcircuit in Equalizer-L..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7259)
  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 7259)
  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: Incoherent feedforward (IFF) plasmid
FEATURES             Location/Qualifiers
     source          1..7259
                     /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      1647..1656
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1653..1655,1656..1658,1659..2372)
                     /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(1653..1655,1656..1658,1659..2369)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1698..1719)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1959..1978)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2306..2327
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           2578..2592
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2620..2748
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(2838..2858)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3211..3228)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3217..3441
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3574..3593)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3725..3772
                     /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             3780..4802
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3780..4802
                     /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    4932..5053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4969..4988)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5023..5042
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5102..5118)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5102..5118)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5115..5137)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5126..5142
                     /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(5150..5156,5157..5174,5175..5180))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5195..5216
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5333..5350)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5504..6092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5584..5603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6263..7054,7055..7123))
                     /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     6886..6905
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7124..7228)
                     /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 accgcttgaa gtctttaatt aaaggcgcca ccatggtgag caagggcgag gagctgttca
     1681 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1741 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1801 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1861 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1921 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1981 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2041 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2101 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     2161 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2221 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     2281 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2341 tcactctcgg catggacgag ctgtacaagt agccgcttga agtctttaat taaaaagctt
     2401 gtgagtatgt gctcgcttcg gcagcacata tactatgtcg aatgaggctt cagtacttta
     2461 cagaatcgtt gcctgcacat cttggaaaca cttgctggga ttacttcttc aggttaaccc
     2521 aacagaaggc tcgagtgctg ttgacagtga gcgccgcttg aagtctttaa ttaaatagtg
     2581 aagccacaga tgtatttaat taaagacttc aagcggtgcc tactgcctcg gagaattcaa
     2641 ggggctactt taggagcaat tatcttgttt actaaaactg aataccttgc tatctctttg
     2701 atacattttt acaaagctga attaaaatgg tataaattaa atcacttttt tcaattgttt
     2761 cctttttttt cctcaggcgg ccgcaatcaa cctctggatt acaaaatttg tgaaagattg
     2821 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     2881 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     2941 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     3001 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     3061 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     3121 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcctcgagt
     3181 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     3241 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3301 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3361 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3421 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     3481 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3541 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3601 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3661 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3721 cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact tccttggcca
     3781 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     3841 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     3901 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     3961 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     4021 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     4081 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     4141 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     4201 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     4261 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     4321 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     4381 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     4441 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     4501 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     4561 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     4621 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     4681 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     4741 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     4801 agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4861 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4921 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4981 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5041 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     5101 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5161 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5221 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5281 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5341 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5401 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5461 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5521 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5581 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5641 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5701 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5761 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5821 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5881 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5941 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6001 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6061 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6121 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6181 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6241 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6301 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6361 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6421 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6481 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6541 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6601 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6661 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6721 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6781 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6841 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6901 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6961 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7021 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7081 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7141 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7201 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
