LOCUS       sequence-354396-        7552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of 3xFLAG-6xHis tagged human GRHL1 (K116A mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7552)
  AUTHORS   Grigolon G, Araldi E, Erni R, Wu JY, Thomas C, La Fortezza M, Laube 
            B, Pohlmann D, Stoffel M, Zarse K, Carreira EM, Ristow M, Fischer F
  TITLE     Grainyhead 1 acts as a drug-inducible conserved transcriptional 
            regulator linked to insulin signaling and lifespan.
  JOURNAL   Nat Commun. 2022 Jan 10;13(1):107. doi: 10.1038/s41467-021-27732-4.
  PUBMED    35013237
REFERENCE   2  (bases 1 to 7552)
  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: "2022-01-10"; volume: "13(1):107. doi"; pages: " 
            10.1038/s41467-021-27732-4"
COMMENT     Sequence Label: pcDNA3.1_Hygro(+)_GRHL1-K116A-L3F6H
FEATURES             Location/Qualifiers
     source          1..7552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        153..532
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        533..736
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     687..707
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     781..800
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        781..799
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2712..2777
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2778..2795
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2892..2909)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2898..3122
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3168..3596
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3255..3274)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3465..3486
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3605..3625)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3610..3939
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3790..3925
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3852..3871
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3988..5013
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3988..5013
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5143..5264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5180..5199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5234..5253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5313..5329)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5313..5329)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5326..5348)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5337..5353
                     /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(5361..5367,5368..5385,5386..5391))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5406..5427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5544..5561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5715..6303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5795..5814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6474..7265,7266..7334))
                     /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     7097..7116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7335..7439)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7514..7533)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
       61 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
      121 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
      181 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      241 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      301 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      361 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      421 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      481 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      541 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      601 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
      661 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
      721 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
      781 taatacgact cactataggg agacccaagc tggctagcat gacacaggag tacgacaaca
      841 aacggccagt gttggttctt cagaatgaag cactttatcc acagcggcgg tcctacacta
      901 gtgaggatga ggcctggaaa tccttcctgg aaaaccctct cactgcagcg accaaagcga
      961 tgatgagcat caatggagat gaagacagcg ccgctgcgct gggcctgctc tatgactact
     1021 acaaggttcc aagagagaga aggtcatcaa cagcaaagcc agaggtggag caccctgagc
     1081 cagatcacag caaaagaaac agcataccaa ttgtgacaga gcagcccctc atctctgctg
     1141 gagaaaacag agtgcaagta ctggccaatg tgccatttaa cattgtcctt ccccatggca
     1201 accagctggg cattgataag agaggccatc tgacagctcc agatacgaca gtcactgtct
     1261 ccatagcaac gatgcctacc cactccatca agacagaaac ccagccacat ggcttcgctg
     1321 tgggaatccc cccagcagtg tatcatcctg agcccactga gcgggtggtg gttttcgatc
     1381 ggaatctcaa tactgaccag ttcagctctg gtgctcaagc cccaaatgct caaaggcgaa
     1441 ctccagactc gaccttctca gagaccttca aggaaggcgt tcaggaggtt ttcttcccct
     1501 cggatctcag tctgcggatg cctggcatga attcagagga ctatgttttt gacagtgttt
     1561 ctgggaacaa ctttgaatat accctagaag cttcaaaatc acttcgacag aagccaggag
     1621 acagtaccat gacgtacctg aacaaaggcc agttctatcc catcaccttg aaggaggtga
     1681 gcagcagtga aggaatccat catcccatca gcaaagttcg aagtgtgatc atggtggttt
     1741 ttgctgaaga caaaagcaga gaagatcagt taaggcattg gaagtactgg cactcccggc
     1801 agcacaccgc taaacaaaga tgcattgaca tagctgacta taaagaaagc ttcaacacta
     1861 tcagtaacgt cgaggagatt gcgtataacg ccatttcctt cacatgggac atcaacgatg
     1921 aagcaaaggt tttcatctct gtgaactgct taagcacaga tttctcttcc cagaagggag
     1981 tgaaggggtt gcctcttaac attcaaattg atacctatag ttacaacaac cgcagcaaca
     2041 agcctgtgca ccgggcctac tgccagatca aggtcttctg tgacaaggga gctgagcgga
     2101 aaatcaggga tgaagaacga aagcaaagca aaagaaaagt ttctgatgtt aaagtgccac
     2161 tgcttccctc tcacaagcga atggatatca cagttttcaa acccttcatt gatctcgata
     2221 ctcagcctgt cctcttcatt cctgacgtgc actttgccaa cttgcagcgg ggcactcatg
     2281 tccttcccat tgcctctgaa gaattggagg gtgaaggctc tgtcttgaaa agggggccgt
     2341 acggcacaga agatgacttt gctgtccctc cttctaccaa gctggcccgg atagaagaac
     2401 caaagagagt gctgctctac gttcgaaagg agtcagaaga agtctttgat gccctgatgc
     2461 tcaaaacccc atctttgaag ggcttgatgg aagctatctc agacaaatac gatgttcccc
     2521 atgacaagat tgggaaaata ttcaagaagt gtaaaaaggg gatcctggtg aacatggacg
     2581 acaacattgt gaagcattac tccaatgagg acaccttcca gctgcagatt gaagaagccg
     2641 gggggtctta caagctcacc ctgacggaga tcggtaccgg cggcggcggc ggcagcggcg
     2701 gcggcggcag cgactacaag gaccacgacg gcgactacaa ggaccacgac atcgactaca
     2761 aggacgacga cgacaagcac caccaccacc accactaagg atccactagt ccagtgtggt
     2821 ggaattctgc agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac
     2881 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2941 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3001 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3061 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3121 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3181 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3241 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3301 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3361 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3421 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3481 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3541 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3601 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3661 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3721 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3781 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3841 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3901 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3961 tccattttcg gatctgatca gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc
     4021 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc
     4081 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     4141 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     4201 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc
     4261 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     4321 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     4381 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     4441 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     4501 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     4561 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     4621 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     4681 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     4741 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     4801 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     4861 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     4921 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     4981 cgccccagca ctcgtccgag ggcaaaggaa tagcacgtgc tacgagattt cgattccacc
     5041 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5101 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     5161 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5221 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     5281 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5341 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5401 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5461 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5521 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5581 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5641 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5701 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5761 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5821 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5881 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5941 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6001 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6061 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6121 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6181 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6241 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6301 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6361 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6421 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6481 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6541 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6601 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6661 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6721 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6781 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6841 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6901 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6961 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7021 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7081 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7141 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7201 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7261 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7321 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7381 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7441 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     7501 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ct
//
