LOCUS       sequence-354551-        7552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of 3xFLAG-6xHis tagged human GRHL1 (T139A 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-T139A-L3F6H
FEATURES             Location/Qualifiers
     source          1..7552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        152..531
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        532..735
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     686..706
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     780..799
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        780..798
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2711..2776
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2777..2794
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2891..2908)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2897..3121
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3167..3595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3254..3273)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3464..3485
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3604..3624)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3609..3938
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3789..3924
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3851..3870
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3987..5012
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3987..5012
                     /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    5142..5263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5179..5198)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5233..5252
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5312..5328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5312..5328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5325..5347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5336..5352
                     /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(5360..5366,5367..5384,5385..5390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5405..5426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5543..5560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5714..6302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5794..5813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6473..7264,7265..7333))
                     /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     7096..7115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7334..7438)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7513..7532)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
       61 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
      121 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      241 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      301 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      361 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      421 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      481 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      541 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      601 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      661 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      721 tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt
      781 aatacgactc actataggga gacccaagct ggctagcatg acacaggagt acgacaacaa
      841 acggccagtg ttggttcttc agaatgaagc actttatcca cagcggcggt cctacactag
      901 tgaggatgag gcctggaaat ccttcctgga aaaccctctc actgcagcga ccaaagcgat
      961 gatgagcatc aatggagatg aagacagcgc cgctgcgctg ggcctgctct atgactacta
     1021 caaggttcca agagagagaa ggtcatcaac agcaaagcca gaggtggagc accctgagcc
     1081 agatcacagc aaaagaaaca gcataccaat tgtgacagag cagcccctca tctctgctgg
     1141 agaaaacaga gtgcaagtac tgaaaaatgt gccatttaac attgtccttc cccatggcaa
     1201 ccagctgggc attgataaga gaggccatct ggccgctcca gatacgacag tcactgtctc
     1261 catagcaacg atgcctaccc actccatcaa gacagaaacc cagccacatg gcttcgctgt
     1321 gggaatcccc ccagcagtgt atcatcctga gcccactgag cgggtggtgg ttttcgatcg
     1381 gaatctcaat actgaccagt tcagctctgg tgctcaagcc ccaaatgctc aaaggcgaac
     1441 tccagactcg accttctcag agaccttcaa ggaaggcgtt caggaggttt tcttcccctc
     1501 ggatctcagt ctgcggatgc ctggcatgaa ttcagaggac tatgtttttg acagtgtttc
     1561 tgggaacaac tttgaatata ccctagaagc ttcaaaatca cttcgacaga agccaggaga
     1621 cagtaccatg acgtacctga acaaaggcca gttctatccc atcaccttga aggaggtgag
     1681 cagcagtgaa ggaatccatc atcccatcag caaagttcga agtgtgatca tggtggtttt
     1741 tgctgaagac aaaagcagag aagatcagtt aaggcattgg aagtactggc actcccggca
     1801 gcacaccgct aaacaaagat gcattgacat agctgactat aaagaaagct tcaacactat
     1861 cagtaacgtc gaggagattg cgtataacgc catttccttc acatgggaca tcaacgatga
     1921 agcaaaggtt ttcatctctg tgaactgctt aagcacagat ttctcttccc agaagggagt
     1981 gaaggggttg cctcttaaca ttcaaattga tacctatagt tacaacaacc gcagcaacaa
     2041 gcctgtgcac cgggcctact gccagatcaa ggtcttctgt gacaagggag ctgagcggaa
     2101 aatcagggat gaagaacgaa agcaaagcaa aagaaaagtt tctgatgtta aagtgccact
     2161 gcttccctct cacaagcgaa tggatatcac agttttcaaa cccttcattg atctcgatac
     2221 tcagcctgtc ctcttcattc ctgacgtgca ctttgccaac ttgcagcggg gcactcatgt
     2281 ccttcccatt gcctctgaag aattggaggg tgaaggctct gtcttgaaaa gggggccgta
     2341 cggcacagaa gatgactttg ctgtccctcc ttctaccaag ctggcccgga tagaagaacc
     2401 aaagagagtg ctgctctacg ttcgaaagga gtcagaagaa gtctttgatg ccctgatgct
     2461 caaaacccca tctttgaagg gcttgatgga agctatctca gacaaatacg atgttcccca
     2521 tgacaagatt gggaaaatat tcaagaagtg taaaaagggg atcctggtga acatggacga
     2581 caacattgtg aagcattact ccaatgagga caccttccag ctgcagattg aagaagccgg
     2641 ggggtcttac aagctcaccc tgacggagat cggtaccggc ggcggcggcg gcagcggcgg
     2701 cggcggcagc gactacaagg accacgacgg cgactacaag gaccacgaca tcgactacaa
     2761 ggacgacgac gacaagcacc accaccacca ccactaagga tccactagtc cagtgtggtg
     2821 gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc
     2881 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2941 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3001 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3061 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3121 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3181 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3241 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3301 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3361 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3421 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3481 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3541 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3601 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3661 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3721 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3781 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3841 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3901 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3961 ccattttcgg atctgatcag cacgtgatga aaaagcctga actcaccgcg acgtctgtcg
     4021 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
     4081 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     4141 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     4201 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct
     4261 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     4321 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     4381 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     4441 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     4501 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     4561 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     4621 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     4681 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     4741 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4801 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4861 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4921 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     4981 gccccagcac tcgtccgagg gcaaaggaat agcacgtgct acgagatttc gattccaccg
     5041 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5101 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     5161 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5221 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     5281 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5341 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5401 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5461 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5521 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5581 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5641 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5701 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5761 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5821 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5881 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5941 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6001 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6061 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6121 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6181 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6241 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6301 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6361 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6421 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6481 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6541 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6601 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6661 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6721 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6781 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6841 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6901 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6961 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7021 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7081 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7141 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7201 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7261 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7321 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7381 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7441 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     7501 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tg
//
