LOCUS       sequence-449283-        6793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mamalian cell expression of SNAP-tag2 in the mitochondrial matrix 
            and cytosolic EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6793)
  AUTHORS   Kuhn S, Nasufovic V, Wilhelm J, Kompa J, de Lange EMF, Lin YH, 
            Egoldt C, Fischer J, Lennoi A, Tarnawski M, Reinstein J, Vlijm R, 
            Hiblot J, Johnsson K
  TITLE     SNAP-tag2 for faster and brighter protein labeling.
  JOURNAL   Nat Chem Biol. 2025 Jul 3. doi: 10.1038/s41589-025-01942-z.
  PUBMED    40610720
REFERENCE   2  (bases 1 to 6793)
  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: "Nat Chem 
            Biol. 2025 Jul 3. doi: 10.1038/s41589-025-01942-z."
COMMENT     Sequence Label: pcDNA5/FRT/TO_Cox8a(4x)-SNAP-tag2-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(86..105)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      899..1487
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1388..1407
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1641..1658
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1775..1796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1811..1816,1817..1834,1835..1841)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1849..1865
                     /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     1854..1876
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1873..1889
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1873..1889
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1938..2059
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1949..1968)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2003..2022
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2189..3211)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(2189..3211)
                     /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(3219..3266)
                     /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     3398..3417
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3550..3774
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3763..3780
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(3824..4537,4538..4540))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(3827..4537,4538..4540))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3869..3890)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4218..4237
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4477..4498
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(4541..4594)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(5153..5224)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             complement(5237..5308)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             complement(5321..5392)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="SVLTPLLLRGLTGSARRLPVPRAK"
     CDS             complement(5405..5479)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     primer_bind     complement(5632..5656)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5666..5684
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5687..5705
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(5707..5910)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5736..5756)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5911..6290
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6462..6481
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6556..6660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6661..6729,6730..6793,1..728)
                     /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 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
       61 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
      121 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
      181 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
      241 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
      301 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
      361 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
      421 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
      481 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
      541 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
      601 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
      661 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
      721 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
      781 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
      841 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
      901 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
      961 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1021 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     1081 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1141 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1201 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1261 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1321 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1381 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1441 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1501 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     1561 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     1621 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     1681 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     1741 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     1801 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     1861 aacaatttca cacaggaaac agctatgacc atgattacgc caagctctag ctagaggtcg
     1921 acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     1981 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     2041 aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc ccgcgctgga
     2101 ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg
     2161 cggtggaatc gaaatctcgt agtacgtgct attcctttgc cctcggacga gtgctggggc
     2221 gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg gccgcgcttc
     2281 tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc
     2341 gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga tagagttggt
     2401 caagaccaat gcggagcata tacgcccgga gccgcggcga tcctgcaagc tccggatgcc
     2461 tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga
     2521 tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg
     2581 ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc
     2641 ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg
     2701 cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc
     2761 atatgaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc
     2821 cgctcgtctg gctaagatcg gccgcagcga tcgcatccat ggcctccgcg accggctgca
     2881 gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg
     2941 agatgcaata ggtcaggctc tcgctgaatt ccccaatgtc aagcacttcc ggaatcggga
     3001 gcgcggccga tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc
     3061 tatttacccg caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt
     3121 cgccctccga gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct
     3181 cgacagacgt cgcggtgagt tcaggctttt tggccaagga agttcctata ctttctagag
     3241 aataggaact tcggaatagg aacttctagg tacgtgaacc atcaccctaa tcaagttttt
     3301 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     3361 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     3421 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     3481 ttaatgcgcc gctacagggc gcgtggggat accccctaga gccccagctg gttctttccg
     3541 cctcagaagc catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct
     3601 cccccttgct gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat
     3661 gcgatgcaat ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca
     3721 aggaaggcac gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg
     3781 ctgatcagcg ggtttaaacg ggccctctag actcgagcgg ttactacttg tacagctcgt
     3841 ccatgccgag agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct
     3901 tctcgttggg gtctttgctc agggcggact gggtgctcag gtagtggttg tcgggcagca
     3961 gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc
     4021 cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg
     4081 ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc
     4141 cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga
     4201 acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct
     4261 ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt
     4321 agcggctgaa gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg
     4381 gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct
     4441 cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga
     4501 tgggcaccac cccggtgaac agctcctcgc ccttgctcac tgggccagga ttctcctcga
     4561 cgtcaccgca tgttagcaga cttcctctgc cctctccact gccgcggccg cccaggcctg
     4621 gtttacccag acggtgacct tcgtgcgcca gcagccattc tttcacgtac agagggcctt
     4681 cgtagccacc tacgtcgccg tcgccctgaa ccacacggtg gcacgggatc agaattggca
     4741 ccggattacc acgcagagcg gttttcaccg ccgcagttgc cgctgggttg ccagccagcg
     4801 ccgccagctg ggaataggag atcacctcgc cgaatttcac tactttcagc agtttccaca
     4861 gtacctgacg ggtaaaggat tcctgctgaa ataccgggtg atgcagggcc ggtacaggga
     4921 actcttcaat cgcctctggc tggtgaaaat atgcgttcag ccatgcggta gcctgcatca
     4981 gcggctccgg gcccggagga ccctgctcgc cctgaccatt gcccacgaaa tagatgcggt
     5041 gcaggccctg ctcgcaaccg ctcagcagca gtttacccag cgggctgtca taggtagtgc
     5101 gtttcatttc gcagtcttta tcgcttccac cagaacctcc gagagagtgg atcttcgctc
     5161 gtggcactgg aagtcggcgg gccgaacctg tcagaccccg caggaggagt ggcgtcagga
     5221 cggacaacga atggatcttt gcgcgcggca ctgggagtcg tcgagcagat cctgtcaagc
     5281 cccgcagcag cagtggcgta aggacagaga gcgaatggat tttagcacgt ggaactggaa
     5341 gccgtctagc agaaccagtc aatccccgca acagcaacgg cgtaagcacg gacaaagaat
     5401 gaattttagc gcgtggcacc gggaggcgcc gggcggaacc tgtcaaacct cgcagcaaca
     5461 gtggcgtgag gaccgacatg gtggttgctg gatatctgca gaattccacc acactggact
     5521 agtggatccg agctcggtac caagcttaag tttaaacgct agagtccgga ggctggatcg
     5581 gtcccggtgt cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg
     5641 gttcactaaa cgagctcgtc gacgatctct atcactgata gggagatctc tatcactgat
     5701 agggagagct ctgcttatat agacctccca ccgtacacgc ctaccgccca tttgcgtcaa
     5761 tggggcggag ttgttacgac attttggaaa gtcccgttga ttttggtgcc aaaacaaact
     5821 cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc gctatccacg
     5881 cccattgatg tactgccaaa accgcatcac catggtaata gcgatgacta atacgtagat
     5941 gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca
     6001 tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc
     6061 caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg
     6121 cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca
     6181 gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac
     6241 taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc aacgcgtata
     6301 tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca
     6361 attgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac
     6421 ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt
     6481 actgagagtg caccataggg gatcgggaga tctcccgatc cgtcgacgtc aggtggcact
     6541 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6601 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     6661 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     6721 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     6781 cgagtgggtt aca
//
