LOCUS       sequence-441396-        6621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MAC3-tagged GFP-Myr (N-terminal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6621)
  AUTHORS   Liu X, Salokas K, Keskitalo S, Martinez-Botia P, Varjosalo M
  TITLE     Analyzing Protein Interactions by MAC-Tag Approaches.
  JOURNAL   Methods Mol Biol. 2023;2690:281-297. doi: 
            10.1007/978-1-0716-3327-4_24.
  PUBMED    37450155
REFERENCE   2  (bases 1 to 6621)
  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: "Methods Mol
            Biol."; date: "2023"; volume: "2690:281-297. doi"; pages: " 
            10.1007/978-1-0716-3327-4_24"
COMMENT     Sequence Label: MAC3-N-GFP-Myr
FEATURES             Location/Qualifiers
     source          1..6621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(10..57)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     189..208
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    341..565
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     554..571
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     protein_bind    642..666
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(668..1378,1379..1381,1382..1384))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELFK"
     primer_bind     complement(710..731)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1059..1078
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1318..1339
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(1390..1414)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1427..1450)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(1487..1510)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(1541..1567)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2251..2275)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2285..2303
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2306..2324
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(2326..2529)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2355..2375)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2530..2909
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3081..3100
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3175..3279
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3280..3348,3349..4140)
                     /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     complement(3498..3517)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4311..4899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4800..4819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5053..5070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5187..5208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5223..5228,5229..5246,5247..5253)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5261..5277
                     /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)."
     primer_bind     5266..5288
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5285..5301
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5285..5301
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5350..5471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5361..5380)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5415..5434
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5601..6621,1..2))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(join(5601..6621,1..2))
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
ORIGIN
        1 ttggccaagg aagttcctat actttctaga gaataggaac ttcggaatag gaacttctag
       61 gtacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
      121 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
      181 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
      241 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga
      301 taccccctag agccccagct ggttctttcc gcctcagaag ccatagagcc caccgcatcc
      361 ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caccccaccc
      421 cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa
      481 aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca
      541 gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac gggggcgcgc
      601 cctactggcg tttgctgcag catttgttgc ccataccggt caccactttg tacaagaaag
      661 ctgggtcctt gaacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca
      721 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca
      781 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
      841 ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga
      901 tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca
      961 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt
     1021 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct
     1081 tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt
     1141 gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca
     1201 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct
     1261 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     1321 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca
     1381 ccatgttgaa gcctgctttt ttgtacaaac ttgtgatatc ggccgctttt tcgaactgcg
     1441 ggtggctcca cgatccacct cccgatccac ctccggaacc tccacctttc tcgaactgcg
     1501 ggtggctcca gctagccatg gtggatccga gctcggtacc ggcatagtca ggaacatcgt
     1561 atgggtagtc cttctccttg aacttcttca ggttctcgct gattctcttc agcaccttca
     1621 gcagcacctc ctttctgtcc cagtccttgc cggtgatctc gtacagggtg gtggctctgt
     1681 ccttgatctc ctcggggatc tctctctggt tcacgttgat gccgatgccc acgatcagct
     1741 tgtccttgct cagctcgcac agcacgccgc tcaccttctt ctcctggaag tacacgtcgt
     1801 tgggccactt caggctgaag gggatctcgg tgatctcctc cagggcctcg ctcacgctca
     1861 ggcccagcac caggggcagc tgcagcaggt tctcgaactc cttggggttc agcaggaagc
     1921 tgaagtacag gccgccctcc tggctcagcc actttctgcc cgggccgcct ctgcccttgg
     1981 tctgtctgtc ggccaccagg gcggtgccgt aggacacgtt ccactccttc agtctctcct
     2041 gggtgctgtc cacctccttc agccagatca ggttcttgaa catggtctcg agcggccgcc
     2101 actgtgctgg atatctgcag aattccacca cactggacta gtggatccga gctcggtacc
     2161 aagcttaagt ttaaacgcta gagtccggag gctggatcgg tcccggtgtc ttctatggag
     2221 gtcaaaacag cgtggatggc gtctccaggc gatctgacgg ttcactaaac gagctcgtcg
     2281 acgatctcta tcactgatag ggagatctct atcactgata gggagagctc tgcttatata
     2341 gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca
     2401 ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg
     2461 gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa
     2521 ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
     2581 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     2641 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     2701 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
     2761 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     2821 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     2881 ctattaataa ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag
     2941 cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca agccttgcct
     3001 tgttgtagct taaattttgc tcgcgcacta ctcagcgacc tccaacacac aagcagggag
     3061 cagatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagggg
     3121 atcgggagat ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3181 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3241 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3301 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3361 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3421 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3481 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3541 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3601 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3661 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3721 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3781 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3841 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3901 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3961 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4021 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4081 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4141 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4201 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4261 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4321 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4381 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4441 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4501 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4561 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4621 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4681 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4741 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4801 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4861 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4921 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4981 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5041 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     5101 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     5161 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     5221 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     5281 cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt cgacggtata
     5341 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     5401 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     5461 ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc
     5521 cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa
     5581 tcgaaatctc gtagtacgtg ctattccttt gccctcggac gagtgctggg gcgtcggttt
     5641 ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgcgggcg
     5701 atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc
     5761 gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg gtcaagacca
     5821 atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg cctccgctcg
     5881 aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa gatgttggcg
     5941 acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg
     6001 ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg
     6061 gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga cgcactgacg
     6121 gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc gcatatgaaa
     6181 tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa cccgctcgtc
     6241 tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg cagaacagcg
     6301 ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa
     6361 taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc
     6421 gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca gctatttacc
     6481 cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc ttcgccctcc
     6541 gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt ctcgacagac
     6601 gtcgcggtga gttcaggctt t
//
