LOCUS       sequence-313536-        7343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a lysosome tagret fusion protein of the pH resistant 
            proteins mCRISPRed and mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7343)
  AUTHORS   Erdogan M, Fabritius A, Basquin J, Griesbeck O
  TITLE     Targeted In Situ Protein Diversification and Intra-organelle 
            Validation in Mammalian Cells.
  JOURNAL   Cell Chem Biol. 2020 May 21;27(5):610-621.e5. doi: 
            10.1016/j.chembiol.2020.02.004. Epub 2020 Mar 5.
  PUBMED    32142629
REFERENCE   2  (bases 1 to 7343)
  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: 
            "10.1016/j.chembiol.2020.02.004"; journalName: "Cell Chem Biol"; 
            date: "2020-05-21- 21"; volume: "27"; issue: "5"; pages: 
            "610-621.e5"
COMMENT     Sequence Label: FRT LAMP3 mCRISPRed_mTagBFP2 pcDNA5
FEATURES             Location/Qualifiers
     source          1..7343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..46)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(200..788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(280..299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(959..1750,1751..1819))
                     /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     1582..1601
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1820..1924)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1999..2018)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2190..2569
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2570..2773
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2724..2744
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2818..2837
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2818..2836
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2869..2877,2878..2892)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      2899..2908
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3802..4509
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             4519..4575
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4576..5175
                     /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(4576..4595)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5072..5092
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5188..5205)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5250..5267)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5256..5480
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5613..5632)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5764..5811
                     /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             5819..6841
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5819..6841
                     /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    6971..7092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7008..7027)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7062..7081
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7141..7157)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7141..7157)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7154..7176)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7165..7181
                     /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(7189..7195,7196..7213,7214..7219))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7234..7255
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
       61 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
      121 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
      181 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      241 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      301 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      361 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      421 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      481 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      541 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      601 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
      661 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      721 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      781 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      841 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
      901 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
      961 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     1021 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     1081 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     1141 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1201 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1261 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1321 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1381 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     1441 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     1501 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     1561 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     1621 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     1681 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     1741 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     1801 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     1861 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     1921 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     1981 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     2041 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     2101 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     2161 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     2221 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     2281 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     2341 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     2401 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     2461 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     2521 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     2581 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     2641 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     2701 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     2761 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     2821 tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagga tctgtacgac
     2881 gatgacgata aggatccggc caccatggcg gtggaaggag gaatgaagtg tgtcaagttt
     2941 ttgctctacg ttctcctgct ggccttctgc gcctgtgcag tgggattgat cgccattggt
     3001 gtagcggttc aggttgtctt gaagcaggcc gaagcggatc tagtgtctaa gggcgaagag
     3061 ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag gttcggtcaa cggccaccaa
     3121 ttcaaatgca caggtgaagg agaaggccgc ccgtacgaag gagtgcaaac catgaggatc
     3181 aaagtcatcg agggaggacc cctgccattt gcctttgaca ttcttgccac gtcgttcatg
     3241 tatggcagcc gtacttttat caagtacccg gccgatattc ctgatttctt taaacagtcc
     3301 tttcctgagg gttttacttg ggaaagagtt acgagatacg aagatggtgg agtcgtcacc
     3361 gtcacccagg acaccagcct tgaggatggc gaactcgttt acaacgtcaa agtcagaggg
     3421 gtaaactttc cctccaatgg tcccgtgatg cagaagaaga ccaagggctg ggaggccgac
     3481 acagagatga tgtatccagc agatggtggt ctgaggggat accttgatcg ggcactgaaa
     3541 gttgatggtg gtggccatct gcattgcaac ttcgtaacaa cttacaggtc aaaaaagacc
     3601 gtcggggaca tcaagatgcc cggtgtccat gccgttgatc accgcctgga aaggatcgag
     3661 gaaagtgaca atgaaaccta cgtagtacaa cgcgaagtcg cagttgccaa gtacagcaac
     3721 cttggtggtg ggatggacga gctgtacaag gctgaggctg ctgctaagga ggctgctgct
     3781 aaggaggctg ctgctaaggc tgtgtctaag ggcgaagagc tgattaagga gaacatgcac
     3841 atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc
     3901 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct
     3961 ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc
     4021 aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg
     4081 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc
     4141 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc
     4201 cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct
     4261 gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg
     4321 atcgcaaacg ccaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct
     4381 ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac
     4441 gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac
     4501 aagcttaatg gaagcggagc tactaacttc agcctgctga agcaagctgg agacgtggag
     4561 gagaaccctg gacctatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     4621 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     4681 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     4741 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     4801 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     4861 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     4921 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag
     4981 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     5041 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     5101 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     5161 aagcccggtg cctgataata ggaattccta tagtgtcacc taaatgcggc cgctcgagtc
     5221 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     5281 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     5341 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     5401 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     5461 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     5521 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5581 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5641 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5701 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     5761 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     5821 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     5881 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     5941 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     6001 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     6061 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     6121 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     6181 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     6241 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     6301 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     6361 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     6421 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     6481 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     6541 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     6601 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     6661 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     6721 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     6781 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     6841 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     6901 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     6961 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     7021 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     7081 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     7141 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     7201 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     7261 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     7321 cggccaacgc gcggggagag gcg
//
