LOCUS       sequence-441398-        6634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MAC3-tagged GFP-NES (C-terminal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6634)
  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 6634)
  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-C-GFP-NES
FEATURES             Location/Qualifiers
     source          1..6634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      138..726
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     627..646
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     878..895
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1012..1033
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1048..1053,1054..1071,1072..1078)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1086..1102
                     /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     1091..1113
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1110..1126
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1110..1126
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1175..1296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1186..1205)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1240..1259
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1426..2448)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(1426..2448)
                     /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
                     "
     protein_bind    complement(2456..2503)
                     /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     2635..2654
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2787..3011
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3000..3017
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3574..3600)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             complement(3574..3600)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3631..3654)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(3691..3714)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    3761..3785
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3787..4497,4498..4500,4501..4503))
                     /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(3829..3850)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4178..4197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4437..4458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(4509..4533)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4712..4736)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4746..4764
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4767..4785
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(4787..4990)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4816..4836)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4991..5370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5542..5561
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5636..5740
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5741..5809,5810..6601)
                     /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(5959..5978)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
       61 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
      121 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
      181 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      241 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
      301 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      361 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      421 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      481 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      541 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      601 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      661 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      721 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      781 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
      841 tgaggatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      901 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      961 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     1021 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     1081 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     1141 gctctagcta gaggtcgacg gtatacagac atgataagat acattgatga gtttggacaa
     1201 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     1261 ttatttgtaa ccattataag ctgcaataaa caagttgggg tgggcgaaga actccagcat
     1321 gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa
     1381 cctttcatag aaggcggcgg tggaatcgaa atctcgtagt acgtgctatt cctttgccct
     1441 cggacgagtg ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt
     1501 ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg
     1561 gacgattgcg tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa
     1621 gctctgatag agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc
     1681 tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca
     1741 cggcctccag aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct
     1801 ccagtcaatg accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc
     1861 cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag
     1921 agcctgcgcg acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg
     1981 gggatcagca atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg
     2041 tccgaatggg ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatggc
     2101 ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac
     2161 accctgtgca cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag
     2221 cacttccgga atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta
     2281 gaaaccatcg gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct
     2341 gaaagcacga gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt
     2401 ttcgatcaga aacttctcga cagacgtcgc ggtgagttca ggctttttgg ccaaggaagt
     2461 tcctatactt tctagagaat aggaacttcg gaataggaac ttctaggtac gtgaaccatc
     2521 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2581 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     2641 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     2701 caccacaccc gccgcgctta atgcgccgct acagggcgcg tggggatacc ccctagagcc
     2761 ccagctggtt ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct
     2821 attgtcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg
     2881 acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt
     2941 ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact
     3001 agaaggcaca gtcgaggctg atcagcgggt ttaaacgggc cctctagact cgagtcatca
     3061 cttctccttg aacttcttca ggttctcgct gattctcttc agcaccttca gcagcacctc
     3121 ctttctgtcc cagtccttgc cggtgatctc gtacagggtg gtggctctgt ccttgatctc
     3181 ctcggggatc tctctctggt tcacgttgat gccgatgccc acgatcagct tgtccttgct
     3241 cagctcgcac agcacgccgc tcaccttctt ctcctggaag tacacgtcgt tgggccactt
     3301 caggctgaag gggatctcgg tgatctcctc cagggcctcg ctcacgctca ggcccagcac
     3361 caggggcagc tgcagcaggt tctcgaactc cttggggttc agcaggaagc tgaagtacag
     3421 gccgccctcc tggctcagcc actttctgcc cgggccgcct ctgcccttgg tctgtctgtc
     3481 ggccaccagg gcggtgccgt aggacacgtt ccactccttc agtctctcct gggtgctgtc
     3541 cacctccttc agccagatca ggttcttgaa gtcggcgtag tcgggcacgt cgtaggggta
     3601 gctggccatg gtgctgccca gctcggtgcc tttctcaaac tgagggtggg accagcttcc
     3661 tccgccgctt ccgccgccag atcctccgcc cttctcgaac tggggatggc tccaccccac
     3721 tttcttgtac aggctggtga tgtcggcggc cttgatatca accactttgt acaagaaagc
     3781 tgggtccttg aacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag
     3841 caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact gggtgctcag
     3901 gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg
     3961 gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat
     4021 gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag
     4081 cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt
     4141 caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt
     4201 gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg
     4261 ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca gggtggtcac
     4321 gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt
     4381 gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc
     4441 gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac
     4501 catgttgaag cctgcttttt tgtacaaact tgttgatggc gcgcccagat ccagatcttc
     4561 cagcgcttcc agcagcatgg taccggtacc acactggact agtggatccg agctcggtac
     4621 caagcttaag tttaaacgct agagtccgga ggctggatcg gtcccggtgt cttctatgga
     4681 ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg gttcactaaa cgagctcgtc
     4741 gacgatctct atcactgata gggagatctc tatcactgat agggagagct ctgcttatat
     4801 agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac
     4861 attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt
     4921 ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa
     4981 accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt
     5041 cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa
     5101 tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta
     5161 aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg
     5221 tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg
     5281 taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc gtaattgatt
     5341 actattaata actagtcaat aatcaatgtc aacgcgtata tctggcccgt acatcgcgaa
     5401 gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca attgtcggtc aagccttgcc
     5461 ttgttgtagc ttaaattttg ctcgcgcact actcagcgac ctccaacaca caagcaggga
     5521 gcagatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccataggg
     5581 gatcgggaga tctcccgatc cgtcgacgtc aggtggcact tttcggggaa atgtgcgcgg
     5641 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5701 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5761 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5821 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5881 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5941 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6001 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6061 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6121 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6181 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6241 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6301 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6361 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6421 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6481 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6541 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6601 actgtcagac caagtttact catatatact ttag
//
