LOCUS       sequence-441410-        6621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MAC3-tagged GFP-NLS (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-NLS
FEATURES             Location/Qualifiers
     source          1..6621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             277..303
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             334..357
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             394..417
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    430..454
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(460..462,463..465,466..1176)
                     /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(505..526)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(766..785)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1113..1134
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1178..1202)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1273..1290)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1279..1503
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1636..1655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1787..1834
                     /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             1842..2864
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1842..2864
                     /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    2994..3115
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3031..3050)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3085..3104
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3164..3180)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3164..3180)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3177..3199)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3188..3204
                     /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(3212..3218,3219..3236,3237..3242))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3257..3278
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3395..3412)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3566..4154)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3646..3665)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4325..5116,5117..5185))
                     /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     4948..4967
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5186..5290)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5365..5384)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5556..5935
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5936..6139
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6090..6110
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    6141..6159
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6162..6180
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6190..6214
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 ctggaggaga tcaccgagat ccccttcagc ctgaagtggc ccaacgacgt gtacttccag
       61 gagaagaagg tgagcggcgt gctgtgcgag ctgagcaagg acaagctgat cgtgggcatc
      121 ggcatcaacg tgaaccagag agagatcccc gaggagatca aggacagagc caccaccctg
      181 tacgagatca ccggcaagga ctgggacaga aaggaggtgc tgctgaaggt gctgaagaga
      241 atcagcgaga acctgaagaa gttcaaggag aaggactacc catacgatgt tcctgactat
      301 gccggtaccg agctcggatc caccatggct agctggagcc acccgcagtt cgagaaaggt
      361 ggaggttccg gaggtggatc gggaggtgga tcgtggagcc acccgcagtt cgaaaaagcg
      421 gccgatatca caagtttgta caaaaaagca ggcttcaaca tggtgagcaa gggcgaggag
      481 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      541 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
      601 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
      661 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
      721 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
      781 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
      841 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
      901 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
      961 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1021 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     1081 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1141 gccgggatca ctctcggcat ggacgagctg ttcaaggacc cagctttctt gtacaaagtg
     1201 gtgaccggtc cgccgaaaac cacccgcaaa gtggaagatt agggcgcgcc cccgtttaaa
     1261 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     1321 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     1381 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     1441 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     1501 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     1561 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1621 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1681 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1741 acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc
     1801 gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg
     1861 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     1921 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     1981 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     2041 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     2101 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     2161 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     2221 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     2281 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     2341 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     2401 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     2461 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     2521 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     2581 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     2641 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     2701 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     2761 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     2821 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt
     2881 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     2941 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     3001 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3061 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     3121 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     3181 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3241 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3301 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3361 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3421 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3481 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3541 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3601 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3661 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3721 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3781 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3841 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3901 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3961 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4021 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4081 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4141 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4201 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4261 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4321 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4381 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4441 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4501 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4561 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4621 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4681 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4741 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4801 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4861 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4921 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4981 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5041 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5101 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5161 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5221 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5281 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     5341 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     5401 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     5461 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     5521 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     5581 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     5641 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     5701 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     5761 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     5821 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     5881 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     5941 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     6001 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     6061 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     6121 ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca gtgatagaga
     6181 tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
     6241 tgacctccat agaagacacc gggaccgatc cagcctccgg actctagcgt ttaaacttaa
     6301 gcttggtacc gagctcggat ccactagtcc agtgtggtgg aattctgcag atatccagca
     6361 cagtggcggc cgctcgagac catgttcaag aacctgatct ggctgaagga ggtggacagc
     6421 acccaggaga gactgaagga gtggaacgtg tcctacggca ccgccctggt ggccgacaga
     6481 cagaccaagg gcagaggcgg cccgggcaga aagtggctga gccaggaggg cggcctgtac
     6541 ttcagcttcc tgctgaaccc caaggagttc gagaacctgc tgcagctgcc cctggtgctg
     6601 ggcctgagcg tgagcgaggc c
//
