LOCUS       sequence-354372-        7552 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of 3xFLAG-6xHis tagged human GRHL1.
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-L3F6H
FEATURES             Location/Qualifiers
     source          1..7552
                     /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"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2794..2859
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2860..2877
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2974..2991)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2980..3204
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3250..3678
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3337..3356)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3547..3568
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3687..3707)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3692..4021
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3872..4007
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3934..3953
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4070..5095
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4070..5095
                     /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    5225..5346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5262..5281)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5316..5335
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5395..5411)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5395..5411)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5408..5430)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5419..5435
                     /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(5443..5449,5450..5467,5468..5473))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5488..5509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5626..5643)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5797..6385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5877..5896)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6556..7347,7348..7416))
                     /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     7179..7198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7417..7521)
                     /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 gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 atgacacagg agtacgacaa caaacggcca gtgttggttc ttcagaatga agcactttat
      961 ccacagcggc ggtcctacac tagtgaggat gaggcctgga aatccttcct ggaaaaccct
     1021 ctcactgcag cgaccaaagc gatgatgagc atcaatggag atgaagacag cgccgctgcg
     1081 ctgggcctgc tctatgacta ctacaaggtt ccaagagaga gaaggtcatc aacagcaaag
     1141 ccagaggtgg agcaccctga gccagatcac agcaaaagaa acagcatacc aattgtgaca
     1201 gagcagcccc tcatctctgc tggagaaaac agagtgcaag tactgaaaaa tgtgccattt
     1261 aacattgtcc ttccccatgg caaccagctg ggcattgata agagaggcca tctgacagct
     1321 ccagatacga cagtcactgt ctccatagca acgatgccta cccactccat caagacagaa
     1381 acccagccac atggcttcgc tgtgggaatc cccccagcag tgtatcatcc tgagcccact
     1441 gagcgggtgg tggttttcga tcggaatctc aatactgacc agttcagctc tggtgctcaa
     1501 gccccaaatg ctcaaaggcg aactccagac tcgaccttct cagagacctt caaggaaggc
     1561 gttcaggagg ttttcttccc ctcggatctc agtctgcgga tgcctggcat gaattcagag
     1621 gactatgttt ttgacagtgt ttctgggaac aactttgaat ataccctaga agcttcaaaa
     1681 tcacttcgac agaagccagg agacagtacc atgacgtacc tgaacaaagg ccagttctat
     1741 cccatcacct tgaaggaggt gagcagcagt gaaggaatcc atcatcccat cagcaaagtt
     1801 cgaagtgtga tcatggtggt ttttgctgaa gacaaaagca gagaagatca gttaaggcat
     1861 tggaagtact ggcactcccg gcagcacacc gctaaacaaa gatgcattga catagctgac
     1921 tataaagaaa gcttcaacac tatcagtaac gtcgaggaga ttgcgtataa cgccatttcc
     1981 ttcacatggg acatcaacga tgaagcaaag gttttcatct ctgtgaactg cttaagcaca
     2041 gatttctctt cccagaaggg agtgaagggg ttgcctctta acattcaaat tgatacctat
     2101 agttacaaca accgcagcaa caagcctgtg caccgggcct actgccagat caaggtcttc
     2161 tgtgacaagg gagctgagcg gaaaatcagg gatgaagaac gaaagcaaag caaaagaaaa
     2221 gtttctgatg ttaaagtgcc actgcttccc tctcacaagc gaatggatat cacagttttc
     2281 aaacccttca ttgatctcga tactcagcct gtcctcttca ttcctgacgt gcactttgcc
     2341 aacttgcagc ggggcactca tgtccttccc attgcctctg aagaattgga gggtgaaggc
     2401 tctgtcttga aaagggggcc gtacggcaca gaagatgact ttgctgtccc tccttctacc
     2461 aagctggccc ggatagaaga accaaagaga gtgctgctct acgttcgaaa ggagtcagaa
     2521 gaagtctttg atgccctgat gctcaaaacc ccatctttga agggcttgat ggaagctatc
     2581 tcagacaaat acgatgttcc ccatgacaag attgggaaaa tattcaagaa gtgtaaaaag
     2641 gggatcctgg tgaacatgga cgacaacatt gtgaagcatt actccaatga ggacaccttc
     2701 cagctgcaga ttgaagaagc cggggggtct tacaagctca ccctgacgga gatcggtacc
     2761 ggcggcggcg gcggcagcgg cggcggcggc agcgactaca aggaccacga cggcgactac
     2821 aaggaccacg acatcgacta caaggacgac gacgacaagc accaccacca ccaccactaa
     2881 ggatccacta gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgctcg
     2941 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     3001 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3061 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3121 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3181 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     3241 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3301 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3361 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3421 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3481 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3541 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3601 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3661 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3721 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3781 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3841 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3901 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3961 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     4021 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtga tgaaaaagcc
     4081 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     4141 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     4201 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     4261 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     4321 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     4381 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     4441 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     4501 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     4561 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     4621 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     4681 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     4741 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     4801 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     4861 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     4921 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     4981 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     5041 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     5101 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     5161 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5221 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5281 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5341 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5401 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5461 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5521 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5581 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5641 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5701 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5761 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5821 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5881 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5941 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6001 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6061 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6121 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6181 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6241 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6301 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6361 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6421 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6481 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6541 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6601 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6661 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6721 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6781 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6841 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6901 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6961 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7021 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7081 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7141 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7201 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7261 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7321 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7381 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7441 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7501 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
