LOCUS       sequence-441416-        6621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MAC3-tagged GFP-NES (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-NES
FEATURES             Location/Qualifiers
     source          1..6621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(28..51)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(88..111)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(142..168)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(852..876)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    886..904
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    907..925
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(927..1130)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(956..976)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1131..1510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1682..1701
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1776..1880
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1881..1949,1950..2741)
                     /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(2099..2118)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2912..3500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3401..3420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3654..3671
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3788..3809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3824..3829,3830..3847,3848..3854)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3862..3878
                     /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     3867..3889
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3886..3902
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3886..3902
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3951..4072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3962..3981)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4016..4035
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4202..5224)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(4202..5224)
                     /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(5232..5279)
                     /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     5411..5430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5563..5787
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5776..5793
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     protein_bind    5864..5888
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(5890..6600,6601..6603,6604..6606))
                     /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(5932..5953)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6281..6300
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6540..6561
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(join(6612..6621,1..15))
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 tttgtacaaa cttgtgatat cggccgcttt ttcgaactgc gggtggctcc acgatccacc
       61 tcccgatcca cctccggaac ctccaccttt ctcgaactgc gggtggctcc agctagccat
      121 ggtggatccg agctcggtac cggcatagtc aggaacatcg tatgggtagt ccttctcctt
      181 gaacttcttc aggttctcgc tgattctctt cagcaccttc agcagcacct cctttctgtc
      241 ccagtccttg ccggtgatct cgtacagggt ggtggctctg tccttgatct cctcggggat
      301 ctctctctgg ttcacgttga tgccgatgcc cacgatcagc ttgtccttgc tcagctcgca
      361 cagcacgccg ctcaccttct tctcctggaa gtacacgtcg ttgggccact tcaggctgaa
      421 ggggatctcg gtgatctcct ccagggcctc gctcacgctc aggcccagca ccaggggcag
      481 ctgcagcagg ttctcgaact ccttggggtt cagcaggaag ctgaagtaca ggccgccctc
      541 ctggctcagc cactttctgc ccgggccgcc tctgcccttg gtctgtctgt cggccaccag
      601 ggcggtgccg taggacacgt tccactcctt cagtctctcc tgggtgctgt ccacctcctt
      661 cagccagatc aggttcttga acatggtctc gagcggccgc cactgtgctg gatatctgca
      721 gaattccacc acactggact agtggatccg agctcggtac caagcttaag tttaaacgct
      781 agagtccgga ggctggatcg gtcccggtgt cttctatgga ggtcaaaaca gcgtggatgg
      841 cgtctccagg cgatctgacg gttcactaaa cgagctcgtc gacgatctct atcactgata
      901 gggagatctc tatcactgat agggagagct ctgcttatat agacctccca ccgtacacgc
      961 ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga
     1021 ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt
     1081 gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata
     1141 gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg
     1201 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata
     1261 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt
     1321 caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg
     1381 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac
     1441 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat
     1501 aatcaatgtc aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc
     1561 ctaagcagat tcttcatgca attgtcggtc aagccttgcc ttgttgtagc ttaaattttg
     1621 ctcgcgcact actcagcgac ctccaacaca caagcaggga gcagatactg gcttaactat
     1681 gcggcatcag agcagattgt actgagagtg caccataggg gatcgggaga tctcccgatc
     1741 cgtcgacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1801 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1861 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1921 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1981 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2041 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2101 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     2161 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2221 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2281 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2341 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2401 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     2461 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2521 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2581 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2641 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2701 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2761 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2821 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2881 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2941 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3001 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3061 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3121 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3181 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3241 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3301 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3361 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3421 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3481 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3541 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3601 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3661 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     3721 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     3781 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     3841 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     3901 accatgatta cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat
     3961 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     4021 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg
     4081 cgaagaactc cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa
     4141 cgattccgaa gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtagtacgt
     4201 gctattcctt tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt
     4261 ctacacagcc atcggtccag acggccgcgc ttctgcgggc gatttgtgta cgcccgacag
     4321 tcccggctcc ggatcggacg attgcgtcgc atcgaccctg cgcccaagct gcatcatcga
     4381 aattgccgtc aaccaagctc tgatagagtt ggtcaagacc aatgcggagc atatacgccc
     4441 ggagccgcgg cgatcctgca agctccggat gcctccgctc gaagtagcgc gtctgctgct
     4501 ccatacaagc caaccacggc ctccagaaga agatgttggc gacctcgtat tgggaatccc
     4561 cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat
     4621 tgttggagcc gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa
     4681 gcatcagctc atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt
     4741 gccagtgata cacatgggga tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt
     4801 gaccgattcc ttgcggtccg aatgggccga acccgctcgt ctggctaaga tcggccgcag
     4861 cgatcgcatc catggcctcc gcgaccggct gcagaacagc gggcagttcg gtttcaggca
     4921 ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca ataggtcagg ctctcgctga
     4981 attccccaat gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa
     5041 cataacgatc tttgtagaaa ccatcggcgc agctatttac ccgcaggaca tatccacgcc
     5101 ctcctacatc gaagctgaaa gcacgagatt cttcgccctc cgagagctgc atcaggtcgg
     5161 agacgctgtc gaacttttcg atcagaaact tctcgacaga cgtcgcggtg agttcaggct
     5221 ttttggccaa ggaagttcct atactttcta gagaatagga acttcggaat aggaacttct
     5281 aggtacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5341 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     5401 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     5461 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtggg
     5521 gataccccct agagccccag ctggttcttt ccgcctcaga agccatagag cccaccgcat
     5581 ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac
     5641 cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg
     5701 aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa
     5761 cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgggtttaa acgggggcgc
     5821 gccctacaga tccagatctt ccagcgcttc cagcagaccg gtcaccactt tgtacaagaa
     5881 agctgggtcc ttgaacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc
     5941 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct
     6001 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
     6061 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt
     6121 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc
     6181 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
     6241 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc
     6301 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
     6361 gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt
     6421 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag
     6481 cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac
     6541 gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct
     6601 caccatgttg aagcctgctt t
//
