LOCUS       sequence-124850-        8017 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Snap-tag substrates with two different fluorophores (red and green) 
            sequentially to separately label young and old mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8017)
  AUTHORS   Katajisto P, Dohla J, Chaffer CL, Pentinmikko N, Marjanovic N, Iqbal
            S, Zoncu R, Chen W, Weinberg RA, Sabatini DM
  TITLE     Stem cells. Asymmetric apportioning of aged mitochondria between 
            daughter cells is required for stemness.
  JOURNAL   Science. 2015 Apr 17;348(6232):340-3. doi: 10.1126/science.1260384. 
            Epub 2015 Apr 2.
  PUBMED    25837514
REFERENCE   2  (bases 1 to 8017)
  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"; doi: 
            "10.1126/science.1260384"; journalName: "Science"; date: 
            "2015-04-17- 17"; volume: "348"; issue: "6232"; pages: "340-3"
COMMENT     Sequence Label: Snap-Omp25
FEATURES             Location/Qualifiers
     source          1..8017
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        131..334
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     285..305
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             366..911
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for mammalian expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     misc_feature    1100..1217
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1266..1776
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1695..1713
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1798..2397
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1798..1817)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2294..2314
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2525..2758
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2830..2951
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2867..2886)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2921..2940
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2991..3126
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3146..3165)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3147..3165)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3175..3192)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3175..3191)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3184..3206)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3333..3788
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3420..3439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3630..3651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3814..3918
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3919..3987,3988..4779)
                     /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(4137..4156)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4950..5538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5439..5458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5692..5709
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5826..5847
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5862..5867,5868..5885,5886..5892)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5900..5916
                     /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     5905..5927
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5924..5940
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5924..5940
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5959..5979
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5961..5979
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6007..6233
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6234..6414
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6461..6586
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7079..7312
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7497..7541
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     primer_bind     7839..7859
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
       61 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      121 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      181 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      241 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      301 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      361 gcgccatgga caaagattgc gaaatgaaac gtaccaccct ggatagccct ctgggcaagc
      421 tggaactgtc tgggtgcgaa cagggcctgc acgagatcaa gctgctgggc aaaggaacat
      481 ctgccgccga cgccgtggaa gtgcctgccc cagccgccgt gctgggcgga ccagagccac
      541 tgatgcaggc caccgcctgg ctcaacgcct actttcacca gcctgaggcc atcgaggagt
      601 tccctgtgcc agccctgcac cacccagtgt tccagcagga gagctttacc cgccaggtgc
      661 tgtggaaact gctgaaagtg gtgaagttcg gagaggtcat cagctaccag cagctggccg
      721 ccctggccgg caatcccgcc gccaccgccg ccgtgaaaac cgccctgagc ggaaatcccg
      781 tgcccattct gatcccctgc caccgggtgg tgtctagctc tggcgccgtg gggggctacg
      841 agggcgggct cgccgtgaaa gagtggctgc tggcccacga gggccacaga ctgggcaagc
      901 ctgggctggg tcctgcaggt ataggcgcgc caggatccac gcgtgggcga ggcgacggag
      961 agccgagtgg agttcctgta gctgtggtgc tgctgccagt gtttgccctt accctggtag
     1021 cagtttgggc cttcgtgaga taccgaaagc agctctgaga attctcgacc tcgagacaaa
     1081 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     1141 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     1201 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggccgcggc
     1261 tcgagggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc
     1321 tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc
     1381 acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg
     1441 cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga
     1501 caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg
     1561 gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag
     1621 agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg
     1681 ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc
     1741 tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccgga gcttaccatg
     1801 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     1861 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1921 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     1981 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     2041 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     2101 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     2161 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     2221 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     2281 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     2341 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc
     2401 ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc atcggtacct
     2461 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     2521 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
     2581 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     2641 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     2701 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     2761 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     2821 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2881 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2941 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     3001 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3061 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3121 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     3181 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     3241 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     3301 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     3361 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3421 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3481 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3541 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3601 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3661 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3721 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     3781 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3841 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3901 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3961 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4021 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     4081 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4141 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     4201 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     4261 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     4321 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     4381 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     4441 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     4501 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     4561 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     4621 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4681 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4741 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4801 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4861 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4921 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4981 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5041 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5101 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5161 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5221 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5281 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5341 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5401 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5461 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5521 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5581 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5641 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5701 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     5761 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     5821 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     5881 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     5941 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa
     6001 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc
     6061 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt
     6121 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg
     6181 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc
     6241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     6301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     6361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     6421 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     6481 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     6541 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     6601 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     6661 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     6721 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     6781 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     6841 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     6901 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
     6961 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     7021 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     7081 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     7141 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     7201 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     7261 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     7321 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     7381 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     7441 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     7501 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     7561 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     7621 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     7681 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag
     7741 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc
     7801 gatcacgaga ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc
     7861 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     7921 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     7981 ttgcagtttt aaaattatgt tttaaaatgg actatca
//
