LOCUS       sequence-441421-        6587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MAC3-tagged GFP (N-terminal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6587)
  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 6587)
  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
FEATURES             Location/Qualifiers
     source          1..6587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        7..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..590
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     541..561
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    592..610
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    613..631
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     641..665
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1349..1375
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1406..1429
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1466..1489
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    1502..1526
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1532..1534,1535..1537,1538..2050)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(1577..1598)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1838..1857)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2185..2206
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2249..2273)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2311..2328)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2317..2541
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2674..2693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2825..2872
                     /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)."
     CDS             2880..3902
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2880..3902
                     /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
                     "
     polyA_signal    4032..4153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4069..4088)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4123..4142
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4202..4218)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4202..4218)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4215..4237)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4226..4242
                     /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(4250..4256,4257..4274,4275..4280))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4295..4316
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4433..4450)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4604..5192)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4684..4703)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5363..6154,6155..6223))
                     /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     5986..6005
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6224..6328)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6403..6422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      421 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      481 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      541 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
      601 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
      661 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      721 ccagcctccg gactctagcg tttaaactta agcttggtac cgagctcgga tccactagtc
      781 cagtgtggtg gaattctgca gatatccagc acagtggcgg ccgctcgaga ccatgttcaa
      841 gaacctgatc tggctgaagg aggtggacag cacccaggag agactgaagg agtggaacgt
      901 gtcctacggc accgccctgg tggccgacag acagaccaag ggcagaggcg gcccgggcag
      961 aaagtggctg agccaggagg gcggcctgta cttcagcttc ctgctgaacc ccaaggagtt
     1021 cgagaacctg ctgcagctgc ccctggtgct gggcctgagc gtgagcgagg ccctggagga
     1081 gatcaccgag atccccttca gcctgaagtg gcccaacgac gtgtacttcc aggagaagaa
     1141 ggtgagcggc gtgctgtgcg agctgagcaa ggacaagctg atcgtgggca tcggcatcaa
     1201 cgtgaaccag agagagatcc ccgaggagat caaggacaga gccaccaccc tgtacgagat
     1261 caccggcaag gactgggaca gaaaggaggt gctgctgaag gtgctgaaga gaatcagcga
     1321 gaacctgaag aagttcaagg agaaggacta cccatacgat gttcctgact atgccggtac
     1381 cgagctcgga tccaccatgg ctagctggag ccacccgcag ttcgagaaag gtggaggttc
     1441 cggaggtgga tcgggaggtg gatcgtggag ccacccgcag ttcgaaaaag cggccgatat
     1501 cacaagtttg tacaaaaaag caggcttcaa catggtgagc aagggcgagg agctgttcac
     1561 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1621 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1681 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1741 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1801 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1861 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1921 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1981 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     2041 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2101 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2161 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2221 cactctcggc atggacgagc tgacaaggac ccagctttct tgtacaaagt ggtgacgtaa
     2281 gctaggggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     2341 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2401 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2461 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     2521 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2581 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2641 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2701 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2761 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2821 cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact tccttggcca
     2881 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     2941 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     3001 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     3061 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     3121 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     3181 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     3241 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     3301 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     3361 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     3421 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     3481 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     3541 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     3601 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     3661 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     3721 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     3781 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     3841 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     3901 agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3961 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4021 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4081 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4141 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4201 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4261 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4321 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4381 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4441 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4501 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4561 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4621 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4681 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4741 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4801 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4861 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4921 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4981 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5041 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5101 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5161 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5221 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5281 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5341 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5401 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5461 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5521 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5581 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5641 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5701 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5761 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5821 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5881 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5941 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6001 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6061 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6121 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6181 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6241 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6301 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     6361 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     6421 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     6481 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     6541 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatata
//
