LOCUS       sequence-441386-        6636 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of MAC3-tagged GFP-Myr (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 Apr 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-Myr
FEATURES             Location/Qualifiers
     source          1..6636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     119..136
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(693..719)
                     /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(693..719)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(750..773)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(810..833)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    880..904
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(906..1616,1617..1619,1620..1622))
                     /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(948..969)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1297..1316
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1556..1577
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(1628..1652)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1831..1855)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1865..1883
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1886..1904
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(1906..2109)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1935..1955)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2110..2489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2661..2680
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2755..2859
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2860..2928,2929..3720)
                     /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(3078..3097)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3891..4479
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4380..4399
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4633..4650
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4767..4788
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4803..4808,4809..4826,4827..4833)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4841..4857
                     /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     4846..4868
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4865..4881
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4865..4881
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4930..5051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4941..4960)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4995..5014
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5181..6203)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(5181..6203)
                     /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(6211..6258)
                     /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     6390..6409
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    join(6542..6636,1..130)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg
       61 gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta
      121 gaaggcacag tcgaggctga tcagcgggtt taaacgggcc ctctagactc gagtcatcac
      181 ttctccttga acttcttcag gttctcgctg attctcttca gcaccttcag cagcacctcc
      241 tttctgtccc agtccttgcc ggtgatctcg tacagggtgg tggctctgtc cttgatctcc
      301 tcggggatct ctctctggtt cacgttgatg ccgatgccca cgatcagctt gtccttgctc
      361 agctcgcaca gcacgccgct caccttcttc tcctggaagt acacgtcgtt gggccacttc
      421 aggctgaagg ggatctcggt gatctcctcc agggcctcgc tcacgctcag gcccagcacc
      481 aggggcagct gcagcaggtt ctcgaactcc ttggggttca gcaggaagct gaagtacagg
      541 ccgccctcct ggctcagcca ctttctgccc gggccgcctc tgcccttggt ctgtctgtcg
      601 gccaccaggg cggtgccgta ggacacgttc cactccttca gtctctcctg ggtgctgtcc
      661 acctccttca gccagatcag gttcttgaag tcggcgtagt cgggcacgtc gtaggggtag
      721 ctggccatgg tgctgcccag ctcggtgcct ttctcaaact gagggtggga ccagcttcct
      781 ccgccgcttc cgccgccaga tcctccgccc ttctcgaact ggggatggct ccaccccact
      841 ttcttgtaca ggctggtgat gtcggcggcc ttgatatcaa ccactttgta caagaaagct
      901 gggtccttga acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
      961 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg
     1021 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     1081 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     1141 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     1201 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     1261 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     1321 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     1381 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
     1441 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     1501 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     1561 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     1621 atgttgaagc ctgctttttt gtacaaactt gttgatggcg cgccctggcg tttgctgcag
     1681 catttgttgc ccatcatggt accggtacca cactggacta gtggatccga gctcggtacc
     1741 aagcttaagt ttaaacgcta gagtccggag gctggatcgg tcccggtgtc ttctatggag
     1801 gtcaaaacag cgtggatggc gtctccaggc gatctgacgg ttcactaaac gagctcgtcg
     1861 acgatctcta tcactgatag ggagatctct atcactgata gggagagctc tgcttatata
     1921 gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca
     1981 ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg
     2041 gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa
     2101 ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
     2161 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     2221 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     2281 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
     2341 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     2401 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     2461 ctattaataa ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag
     2521 cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca agccttgcct
     2581 tgttgtagct taaattttgc tcgcgcacta ctcagcgacc tccaacacac aagcagggag
     2641 cagatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagggg
     2701 atcgggagat ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2761 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2821 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2881 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2941 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3001 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3061 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3121 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3181 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3241 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3301 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3361 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3421 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3481 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3541 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3601 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3661 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3721 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3781 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3841 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3901 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3961 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4021 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4081 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4141 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4201 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4261 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4321 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4381 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4441 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4501 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4561 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4621 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     4681 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4741 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     4801 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     4861 cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt cgacggtata
     4921 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     4981 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     5041 ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc
     5101 cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa
     5161 tcgaaatctc gtagtacgtg ctattccttt gccctcggac gagtgctggg gcgtcggttt
     5221 ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgcgggcg
     5281 atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc
     5341 gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg gtcaagacca
     5401 atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg cctccgctcg
     5461 aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa gatgttggcg
     5521 acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg
     5581 ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg
     5641 gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga cgcactgacg
     5701 gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc gcatatgaaa
     5761 tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa cccgctcgtc
     5821 tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg cagaacagcg
     5881 ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa
     5941 taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc
     6001 gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca gctatttacc
     6061 cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc ttcgccctcc
     6121 gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt ctcgacagac
     6181 gtcgcggtga gttcaggctt tttggccaag gaagttccta tactttctag agaataggaa
     6241 cttcggaata ggaacttcta ggtacgtgaa ccatcaccct aatcaagttt tttggggtcg
     6301 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     6361 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     6421 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     6481 ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc cgcctcagaa
     6541 gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg
     6601 ctgtcctgcc ccaccccacc ccccagaata gaatga
//
