LOCUS       sequence-354393-        7552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of 3xFLAG-6xHis tagged human GRHL1 (S95A 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-S95A-L3F6H
FEATURES             Location/Qualifiers
     source          1..7552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..62)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     768..788
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     862..881
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        862..880
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2793..2858
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2859..2876
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2973..2990)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2979..3203
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3249..3677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3336..3355)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3546..3567
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3686..3706)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3691..4020
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3871..4006
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3933..3952
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4069..5094
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4069..5094
                     /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    5224..5345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5261..5280)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5315..5334
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5394..5410)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5394..5410)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5407..5429)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5418..5434
                     /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(5442..5448,5449..5466,5467..5472))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5487..5508
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5625..5642)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5796..6384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5876..5895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6555..7346,7347..7415))
                     /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     7178..7197
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7416..7520)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
       61 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      121 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
      181 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      781 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
      841 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagca
      901 tgacacagga gtacgacaac aaacggccag tgttggttct tcagaatgaa gcactttatc
      961 cacagcggcg gtcctacact agtgaggatg aggcctggaa atccttcctg gaaaaccctc
     1021 tcactgcagc gaccaaagcg atgatgagca tcaatggaga tgaagacagc gccgctgcgc
     1081 tgggcctgct ctatgactac tacaaggttc caagagagag aaggtcatca acagcaaagc
     1141 cagaggtgga gcaccctgag ccagatcaca gcaaaagaaa cgccatacca attgtgacag
     1201 agcagcccct catctctgct ggagaaaaca gagtgcaagt actgaaaaat gtgccattta
     1261 acattgtcct tccccatggc aaccagctgg gcattgataa gagaggccat ctgacagctc
     1321 cagatacgac agtcactgtc tccatagcaa cgatgcctac ccactccatc aagacagaaa
     1381 cccagccaca tggcttcgct gtgggaatcc ccccagcagt gtatcatcct gagcccactg
     1441 agcgggtggt ggttttcgat cggaatctca atactgacca gttcagctct ggtgctcaag
     1501 ccccaaatgc tcaaaggcga actccagact cgaccttctc agagaccttc aaggaaggcg
     1561 ttcaggaggt tttcttcccc tcggatctca gtctgcggat gcctggcatg aattcagagg
     1621 actatgtttt tgacagtgtt tctgggaaca actttgaata taccctagaa gcttcaaaat
     1681 cacttcgaca gaagccagga gacagtacca tgacgtacct gaacaaaggc cagttctatc
     1741 ccatcacctt gaaggaggtg agcagcagtg aaggaatcca tcatcccatc agcaaagttc
     1801 gaagtgtgat catggtggtt tttgctgaag acaaaagcag agaagatcag ttaaggcatt
     1861 ggaagtactg gcactcccgg cagcacaccg ctaaacaaag atgcattgac atagctgact
     1921 ataaagaaag cttcaacact atcagtaacg tcgaggagat tgcgtataac gccatttcct
     1981 tcacatggga catcaacgat gaagcaaagg ttttcatctc tgtgaactgc ttaagcacag
     2041 atttctcttc ccagaaggga gtgaaggggt tgcctcttaa cattcaaatt gatacctata
     2101 gttacaacaa ccgcagcaac aagcctgtgc accgggccta ctgccagatc aaggtcttct
     2161 gtgacaaggg agctgagcgg aaaatcaggg atgaagaacg aaagcaaagc aaaagaaaag
     2221 tttctgatgt taaagtgcca ctgcttccct ctcacaagcg aatggatatc acagttttca
     2281 aacccttcat tgatctcgat actcagcctg tcctcttcat tcctgacgtg cactttgcca
     2341 acttgcagcg gggcactcat gtccttccca ttgcctctga agaattggag ggtgaaggct
     2401 ctgtcttgaa aagggggccg tacggcacag aagatgactt tgctgtccct ccttctacca
     2461 agctggcccg gatagaagaa ccaaagagag tgctgctcta cgttcgaaag gagtcagaag
     2521 aagtctttga tgccctgatg ctcaaaaccc catctttgaa gggcttgatg gaagctatct
     2581 cagacaaata cgatgttccc catgacaaga ttgggaaaat attcaagaag tgtaaaaagg
     2641 ggatcctggt gaacatggac gacaacattg tgaagcatta ctccaatgag gacaccttcc
     2701 agctgcagat tgaagaagcc ggggggtctt acaagctcac cctgacggag atcggtaccg
     2761 gcggcggcgg cggcagcggc ggcggcggca gcgactacaa ggaccacgac ggcgactaca
     2821 aggaccacga catcgactac aaggacgacg acgacaagca ccaccaccac caccactaag
     2881 gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc ggccgctcga
     2941 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3001 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3061 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3121 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3181 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3241 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3301 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3361 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3421 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3481 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3541 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3601 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3661 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3721 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3781 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3841 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3901 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3961 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4021 aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgat gaaaaagcct
     4081 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     4141 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     4201 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     4261 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     4321 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     4381 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     4441 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     4501 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     4561 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     4621 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     4681 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     4741 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     4801 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     4861 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     4921 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     4981 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     5041 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgtg
     5101 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     5161 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     5221 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5281 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5341 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     5401 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5461 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5521 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5581 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5641 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5701 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5761 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5821 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5881 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5941 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6001 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6061 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6121 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6181 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6241 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6301 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6361 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6421 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6481 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6541 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6601 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6661 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6721 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6781 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6841 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6901 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6961 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7021 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7081 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7141 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7201 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7261 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7321 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7381 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7441 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7501 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cg
//
