LOCUS       sequence-441395-        6636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MAC3-tagged GFP-NLS (C-terminal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6636)
  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 6636)
  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-NLS
FEATURES             Location/Qualifiers
     source          1..6636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     237..256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(475..579)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(654..673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        845..1224
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1225..1428
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1379..1399
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1430..1448
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1451..1469
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1479..1503
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1682..1706
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1712..1714,1715..1717,1718..2428)
                     /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(1757..1778)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2018..2037)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2365..2386
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2430..2454)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2501..2524
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2561..2584
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2615..2641
                     /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             2615..2641
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3198..3215)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3204..3428
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3561..3580)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3712..3759
                     /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             3767..4789
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3767..4789
                     /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    4919..5040
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4956..4975)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5010..5029
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5089..5105)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5089..5105)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5102..5124)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5113..5129
                     /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(5137..5143,5144..5161,5162..5167))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5182..5203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5320..5337)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5491..6079)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5571..5590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6250..6636,1..405,406..474))
                     /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"
ORIGIN
        1 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
       61 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
      121 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
      181 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
      241 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
      301 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
      361 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
      421 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
      481 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
      541 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
      601 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
      661 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
      721 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      781 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      841 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      901 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      961 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1021 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1081 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1141 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1201 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     1261 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     1321 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     1381 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt
     1441 gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag
     1501 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
     1561 agcctccgga ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca
     1621 gtgtggtacc ggtaccatgc cgccgaaaac cacccgcaaa gtggaagatg gcgcgccatc
     1681 aacaagtttg tacaaaaaag caggcttcaa catggtgagc aagggcgagg agctgttcac
     1741 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1801 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1861 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1921 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1981 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     2041 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     2101 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     2161 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     2221 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2281 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2341 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2401 cactctcggc atggacgagc tgttcaagga cccagctttc ttgtacaaag tggttgatat
     2461 caaggccgcc gacatcacca gcctgtacaa gaaagtgggg tggagccatc cccagttcga
     2521 gaagggcgga ggatctggcg gcggaagcgg cggaggaagc tggtcccacc ctcagtttga
     2581 gaaaggcacc gagctgggca gcaccatggc cagctacccc tacgacgtgc ccgactacgc
     2641 cgacttcaag aacctgatct ggctgaagga ggtggacagc acccaggaga gactgaagga
     2701 gtggaacgtg tcctacggca ccgccctggt ggccgacaga cagaccaagg gcagaggcgg
     2761 cccgggcaga aagtggctga gccaggaggg cggcctgtac ttcagcttcc tgctgaaccc
     2821 caaggagttc gagaacctgc tgcagctgcc cctggtgctg ggcctgagcg tgagcgaggc
     2881 cctggaggag atcaccgaga tccccttcag cctgaagtgg cccaacgacg tgtacttcca
     2941 ggagaagaag gtgagcggcg tgctgtgcga gctgagcaag gacaagctga tcgtgggcat
     3001 cggcatcaac gtgaaccaga gagagatccc cgaggagatc aaggacagag ccaccaccct
     3061 gtacgagatc accggcaagg actgggacag aaaggaggtg ctgctgaagg tgctgaagag
     3121 aatcagcgag aacctgaaga agttcaagga gaagtgatga ctcgagtcta gagggcccgt
     3181 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3241 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3301 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3361 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3421 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3481 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3541 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3601 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3661 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     3721 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     3781 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     3841 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     3901 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     3961 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     4021 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     4081 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     4141 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     4201 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     4261 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     4321 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     4381 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     4441 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     4501 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     4561 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     4621 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     4681 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     4741 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     4801 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4861 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4921 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4981 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5041 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5101 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5161 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5221 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5281 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5341 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5401 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5461 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5521 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5581 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5641 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5701 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5761 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5821 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5881 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5941 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6001 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6061 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6121 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6181 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6241 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6301 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6361 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6421 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6481 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6541 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6601 ggcttcattc agctccggtt cccaacgatc aaggcg
//
