LOCUS       sequence-354567-        7552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of 3xFLAG-6xHis tagged human GRHL1 (S374A 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-S374A-L3F6H
FEATURES             Location/Qualifiers
     source          1..7552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..439
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        440..643
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     594..614
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     688..707
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        688..706
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2619..2684
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2685..2702
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2799..2816)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2805..3029
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3075..3503
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3162..3181)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3372..3393
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3512..3532)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3517..3846
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3697..3832
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3759..3778
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3895..4920
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3895..4920
                     /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    5050..5171
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5087..5106)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5141..5160
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5220..5236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5220..5236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5233..5255)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5244..5260
                     /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(5268..5274,5275..5292,5293..5298))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5313..5334
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5451..5468)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5622..6210)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5702..5721)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6381..7172,7173..7241))
                     /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     7004..7023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7242..7346)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7421..7440)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
       61 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      121 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      181 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      481 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      541 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      601 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      661 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
      721 ctagcatgac acaggagtac gacaacaaac ggccagtgtt ggttcttcag aatgaagcac
      781 tttatccaca gcggcggtcc tacactagtg aggatgaggc ctggaaatcc ttcctggaaa
      841 accctctcac tgcagcgacc aaagcgatga tgagcatcaa tggagatgaa gacagcgccg
      901 ctgcgctggg cctgctctat gactactaca aggttccaag agagagaagg tcatcaacag
      961 caaagccaga ggtggagcac cctgagccag atcacagcaa aagaaacagc ataccaattg
     1021 tgacagagca gcccctcatc tctgctggag aaaacagagt gcaagtactg aaaaatgtgc
     1081 catttaacat tgtccttccc catggcaacc agctgggcat tgataagaga ggccatctga
     1141 cagctccaga tacgacagtc actgtctcca tagcaacgat gcctacccac tccatcaaga
     1201 cagaaaccca gccacatggc ttcgctgtgg gaatcccccc agcagtgtat catcctgagc
     1261 ccactgagcg ggtggtggtt ttcgatcgga atctcaatac tgaccagttc agctctggtg
     1321 ctcaagcccc aaatgctcaa aggcgaactc cagactcgac cttctcagag accttcaagg
     1381 aaggcgttca ggaggttttc ttcccctcgg atctcagtct gcggatgcct ggcatgaatt
     1441 cagaggacta tgtttttgac agtgtttctg ggaacaactt tgaatatacc ctagaagctt
     1501 caaaatcact tcgacagaag ccaggagaca gtaccatgac gtacctgaac aaaggccagt
     1561 tctatcccat caccttgaag gaggtgagca gcagtgaagg aatccatcat cccatcagca
     1621 aagttcgaag tgtgatcatg gtggtttttg ctgaagacaa aagcagagaa gatcagttaa
     1681 ggcattggaa gtactggcac tcccggcagc acaccgctaa acaaagatgc attgacatag
     1741 ctgactataa agaaagcttc aacactatca gtaacgtcga ggagattgcg tataacgcca
     1801 tttccttcac atgggacatc aacgatgaag caaaggtttt catcgccgtg aactgcttaa
     1861 gcacagattt ctcttcccag aagggagtga aggggttgcc tcttaacatt caaattgata
     1921 cctatagtta caacaaccgc agcaacaagc ctgtgcaccg ggcctactgc cagatcaagg
     1981 tcttctgtga caagggagct gagcggaaaa tcagggatga agaacgaaag caaagcaaaa
     2041 gaaaagtttc tgatgttaaa gtgccactgc ttccctctca caagcgaatg gatatcacag
     2101 ttttcaaacc cttcattgat ctcgatactc agcctgtcct cttcattcct gacgtgcact
     2161 ttgccaactt gcagcggggc actcatgtcc ttcccattgc ctctgaagaa ttggagggtg
     2221 aaggctctgt cttgaaaagg gggccgtacg gcacagaaga tgactttgct gtccctcctt
     2281 ctaccaagct ggcccggata gaagaaccaa agagagtgct gctctacgtt cgaaaggagt
     2341 cagaagaagt ctttgatgcc ctgatgctca aaaccccatc tttgaagggc ttgatggaag
     2401 ctatctcaga caaatacgat gttccccatg acaagattgg gaaaatattc aagaagtgta
     2461 aaaaggggat cctggtgaac atggacgaca acattgtgaa gcattactcc aatgaggaca
     2521 ccttccagct gcagattgaa gaagccgggg ggtcttacaa gctcaccctg acggagatcg
     2581 gtaccggcgg cggcggcggc agcggcggcg gcggcagcga ctacaaggac cacgacggcg
     2641 actacaagga ccacgacatc gactacaagg acgacgacga caagcaccac caccaccacc
     2701 actaaggatc cactagtcca gtgtggtgga attctgcaga tatccagcac agtggcggcc
     2761 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     2821 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2881 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2941 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3001 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3061 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3121 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3181 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3241 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3301 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3361 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3421 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3481 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3541 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3601 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3661 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3721 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3781 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3841 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgatgaaa
     3901 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     3961 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     4021 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     4081 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     4141 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     4201 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     4261 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     4321 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     4381 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     4441 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     4501 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     4561 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     4621 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     4681 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     4741 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     4801 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     4861 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     4921 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4981 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5041 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5101 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5161 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5221 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5281 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5341 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5401 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5461 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5521 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5581 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5641 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5701 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5761 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5821 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5881 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5941 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6001 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6061 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6121 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6181 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6241 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6301 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6361 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6421 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6481 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6541 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6601 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6661 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6721 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6781 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6841 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6901 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6961 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7021 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7081 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7141 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7201 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7261 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7321 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     7381 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     7441 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     7501 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aa
//
