LOCUS       sequence-457433-        6015 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of C-terminal fragment of HRAS responsible for 
            farnesylation tagged with GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6015)
  AUTHORS   Winkelmann H, Richter CP, Eising J, Piehler J, Kurre R
  TITLE     Correlative single-molecule and structured illumination microscopy 
            of fast dynamics at the plasma membrane.
  JOURNAL   Nat Commun. 2024 Jul 10;15(1):5813. doi: 10.1038/s41467-024-49876-9.
  PUBMED    38987559
REFERENCE   2  (bases 1 to 6015)
  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: "2024-07-10"; volume: "15(1):5813. doi"; pages: " 
            10.1038/s41467-024-49876-9"
COMMENT     Sequence Label: pSems meGFP-farnesyl
FEATURES             Location/Qualifiers
     source          1..6015
                     /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             join(927..929,930..932,933..1643)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(972..993)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1233..1252)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1580..1601
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          1791..2020
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2003..2022
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2093..2667,2668..2670,2671..2679)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2260..2277)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2487..2506
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2702..3505
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2765..2784)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3375..3394
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3635..3756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3672..3691)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3858..3874)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3858..3874)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3871..3893)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3882..3898
                     /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(3906..3912,3913..3930,3931..3936))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3951..3972
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4089..4106)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4260..4848)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4340..4359)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5019..5810,5811..5879))
                     /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     5642..5661
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5880..5984)
                     /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 gcttggtacc
      901 gagctcggat cgatatcgaa ttcaccatgg tgagcaaggg cgaggagctg ttcaccgggg
      961 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1021 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1081 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1141 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1201 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1261 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1321 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1381 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1441 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1501 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaaactg agcaaagacc
     1561 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1621 tcggcatgga cgagctgtac aagggatcca agctgaaccc tcctgatgag agtggccccg
     1681 gctgcatgag ctgcaagtgt gtgctctcct gactcgagtg aggcggccgc atagataact
     1741 gatccagtgt gctggaatta attcgctgtc tgcgagggcc agctgttggg gtgagtactc
     1801 cctctcaaaa gcgggcatga cttctgcgct aagattgtca gtttccaaaa acgaggagga
     1861 tttgatattc acctggcccg cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga
     1921 aaagacaatc tttttgttgt caagcttgag gtgtggcagg cttgagatct ggccatacac
     1981 ttgagtgaca atgacatcca ctttgccttt ctctccacag gtgtccactc ccaggtccaa
     2041 ctgcaggtcg agcatgcatc tagggcggcc aattccgccc ctctcccccc cccccctctc
     2101 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     2161 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     2221 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     2281 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     2341 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     2401 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     2461 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     2521 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     2581 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     2641 ttttcctttg aaaaacacga tgataagctt gccacaaccc gggataattc ctgcagccaa
     2701 tatgggatcg gccattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2761 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2821 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     2881 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     2941 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3001 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3061 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3121 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3181 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     3241 catgcccgac ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3301 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3361 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3421 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3481 tcgccttctt gacgagttct tctgagggga tcaattctct agataactga tcataatcag
     3541 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     3601 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     3661 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3721 tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtcgagtgca ttctagttgt
     3781 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     3841 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3901 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3961 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4021 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4081 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4141 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4201 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4261 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4321 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4381 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4441 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4501 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4561 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4621 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4681 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4741 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4801 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4861 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4921 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4981 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5041 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5101 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5161 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5221 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5281 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5341 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5401 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5461 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5521 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5581 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5641 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5701 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5761 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5821 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5881 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5941 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6001 gtgccacctg acgtc
//
