LOCUS       sequence-449279-        6898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mamalian cell expression of SNAP-tag2 on mitochondrial surface and 
            cytosolic EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6898)
  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 6898)
  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_TOMM20-SNAP-tag2-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(82..186)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(261..280)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        452..830
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        832..1035
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     986..1006
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1037..1055
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1058..1076
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1086..1110
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2253..2306
                     /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             join(2307..2309,2310..3023)
                     /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             join(2307..2309,2310..3020)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2349..2370)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2610..2629)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2957..2978
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3067..3084)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3073..3297
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3430..3449)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3581..3628
                     /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             3636..4658
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3636..4658
                     /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    4788..4909
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4825..4844)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4879..4898
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4958..4974)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4958..4974)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4971..4993)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4982..4998
                     /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(5006..5012,5013..5030,5031..5036))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5051..5072
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5189..5206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5360..5948)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5440..5459)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6119..6898,1..12,13..81))
                     /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     6742..6761
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
       61 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      121 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      181 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
      241 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
      301 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
      361 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
      421 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      481 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      541 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      601 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      661 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      721 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      781 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat tgtgatgcgg
      841 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      901 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      961 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     1021 tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt
     1081 cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
     1141 ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt
     1201 ggtaccgagc tcggatccac tagtccagtg tggtggaatt ctgcagatat ccagcaacca
     1261 tggtgggtcg gaacagcgcc atcgccgccg gtgtatgcgg ggcccttttc attgggtact
     1321 gcatctactt cgaccgcaaa agacgaagtg accccaactt caagaacagg cttcgagaac
     1381 gaagaaagaa acagaagctt gccaaggaga gagctgggct ttccaagtta cctgacctta
     1441 aagatgctga agctgttcag aagttcttcc ttgaagaaat acagcttggt gaagagttac
     1501 tagctcaagg tgaatatgag aagggcgtag accatctgac aaatgcaatt gctgtgtgtg
     1561 gacagccaca gcagttactg caggtcttac agcaaactct tccaccacca gtgttccaga
     1621 tgcttctgac taagctccca acaattagtc agagaattgt aagtgctcag agcttggctg
     1681 aagatgatgt ggaaggcggt agcggggatc caccggtcgg aggtgataaa gactgcgaaa
     1741 tgaaacgcac tacctatgac agcccgctgg gtaaactgct gctgagcggt tgcgagcagg
     1801 gcctgcaccg catctatttc gtgggcaatg gtcagggcga gcagggtcct ccgggcccgg
     1861 agccgctgat gcaggctacc gcatggctga acgcatattt tcaccagcca gaggcgattg
     1921 aagagttccc tgtaccggcc ctgcatcacc cggtatttca gcaggaatcc tttacccgtc
     1981 aggtactgtg gaaactgctg aaagtagtga aattcggcga ggtgatctcc tattcccagc
     2041 tggcggcgct ggctggcaac ccagcggcaa ctgcggcggt gaaaaccgct ctgcgtggta
     2101 atccggtgcc aattctgatc ccgtgccacc gtgtggttca gggcgacggc gacgtaggtg
     2161 gctacgaagg ccctctgtac gtgaaagaat ggctgctggc gcacgaaggt caccgtctgg
     2221 gtaaaccagg cctgggcggc cgcggcagtg gagagggcag aggaagtctg ctaacatgcg
     2281 gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg
     2341 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2401 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2461 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2521 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2581 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2641 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2701 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2761 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2821 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2881 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2941 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3001 gcatggacga gctgtacaag tagtaaccgc tcgagtctag agggcccgtt taaacccgct
     3061 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3121 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3181 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3241 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     3301 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     3361 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3421 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3481 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3541 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     3601 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     3661 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     3721 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     3781 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     3841 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     3901 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     3961 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     4021 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     4081 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     4141 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     4201 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     4261 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     4321 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     4381 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     4441 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     4501 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     4561 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     4621 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     4681 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4741 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4801 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4861 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4921 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4981 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5041 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5101 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5161 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5221 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5281 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5341 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5401 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5461 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5521 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5581 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5641 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5701 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5761 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5821 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5881 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5941 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6001 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6061 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6121 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6181 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6241 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6301 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6361 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6421 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6481 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6541 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6601 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6661 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6721 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6781 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6841 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccag
//
