LOCUS       sequence-355919-        6997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV overexpression of SNAP-tag fast in the lysosome of mammalian 
            cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6997)
  AUTHORS   Frei MS, Koch B, Hiblot J, Johnsson K
  TITLE     Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic 
            Fluorescent Probes.
  JOURNAL   ACS Chem Biol. 2022 Jun 17;17(6):1321-1327. doi: 
            10.1021/acschembio.2c00041. Epub 2022 May 18.
  PUBMED    35584304
REFERENCE   2  (bases 1 to 6997)
  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: 
            "10.1021/acschembio.2c00041"; journalName: "ACS Chem Biol"; date: 
            "2022-06-17- 17"; volume: "17"; issue: "6"; pages: "1321-1327"
COMMENT     Sequence Label: pcDNA5/FRT/TO_LAMP1_STf
FEATURES             Location/Qualifiers
     source          1..6997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1041..1050
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1043..1126,1127..2293)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     primer_bind     complement(2949..2966)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2955..3179
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3312..3331)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3463..3510
                     /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             3518..4540
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3518..4540
                     /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    4670..4791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4707..4726)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4761..4780
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4840..4856)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4840..4856)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4853..4875)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4864..4880
                     /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(4888..4894,4895..4912,4913..4918))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4933..4954
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5071..5088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5242..5830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5322..5341)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6001..6792,6793..6861))
                     /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     6624..6643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6862..6966)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatccagcaa ccatggcggc ccccggcagc gcccggcgac ccctgctgct
     1081 gctactgctg ttgctgctgc tcggcctcat gcattgtgcg tcagcagcaa tgtttatggt
     1141 gaaaaatggc aacgggaccg cgtgcataat ggccaacttc tctgctgcct tctcagtgaa
     1201 ctacgacacc aagagtggcc ctaagaacat gacctttgac ctgccatcag atgccacagt
     1261 ggtgctcaac cgcagctcct gtggaaaaga gaacacttct gaccccagtc tcgtgattgc
     1321 ttttggaaga ggacatacac tcactctcaa tttcacgaga aatgcaacac gttacagcgt
     1381 ccagctcatg agttttgttt ataacttgtc agacacacac cttttcccca atgcgagctc
     1441 caaagaaatc aagactgtgg aatctataac tgacatcagg gcagatatag ataaaaaata
     1501 cagatgtgtt agtggcaccc aggtccacat gaacaacgtg accgtaacgc tccatgatgc
     1561 caccatccag gcgtaccttt ccaacagcag cttcagcagg ggagagacac gctgtgaaca
     1621 agacaggcct tccccaacca cagcgccccc tgcgccaccc agcccctcgc cctcacccgt
     1681 gcccaagagc ccctctgtgg acaagtacaa cgtgagcggc accaacggga cctgcctgct
     1741 ggccagcatg gggctgcagc tgaacctcac ctatgagagg aaggacaaca cgacggtgac
     1801 aaggcttctc aacatcaacc ccaacaagac ctcggccagc gggagctgcg gcgcccacct
     1861 ggtgactctg gagctgcaca gcgagggcac caccgtcctg ctcttccagt tcgggatgaa
     1921 tgcaagttct agccggtttt tcctacaagg aatccagttg aatacaattc ttcctgacgc
     1981 cagagacccc gcctttaaag ctgccaacgg ctccctgcga gcgctgcagg ccacagtcgg
     2041 caattcctac aagtgcaacg cggaggagca cgtccgtgtc acgaaggcgt tttcagtcaa
     2101 tatattcaaa gtgtgggtcc aggctttcaa ggtggaaggt ggccagtttg gctctgtgga
     2161 ggagtgtctg ctggacgaga acagcatgct gatccccatc gctgtgggtg gtgccctggc
     2221 ggggctggtc ctcatcgtcc tcatcgccta cctcgtcggc aggaagagga gtcacgcagg
     2281 ctaccagact atcggctcca ccggctccac aggctccacc ggcgcggtcg acggtaccgc
     2341 gggcccggga tccatcgcca ccgataaaga ctgcgaaatg aaacgcacta ccctggacag
     2401 cccgctgggt aaactggaac tgagcggttg cgagcagggc ctgcaccgca tcattttcct
     2461 gggcaaaggt acgagcgctg cggatgcggt cgaagtaccg gctccggctg cggttctggg
     2521 tggcccggag ccgctgatgc aggctaccgc atggctgaac gcatattttc accagccaga
     2581 ggcgattgaa gagttccctg taccggccct gcatcacccg gtatttcagc aggaatcctt
     2641 tacccgtcag gtactgtgga aactgctgaa agtagtgaaa ttcggcgagg tgatctccta
     2701 ttcccatctg gcggcgctgg ctggcaaccc agcggcaact gcggcggtga aaaccgctct
     2761 gtctggtaat ccggtgccaa ttctgatccc gtgccaccgt gtggttcagg gcgacctgga
     2821 cgtaggtggc tacgaaggcg gcctggctgt gaaagaatgg ctgctggcgc acgaaggtca
     2881 ccgtctgggt aaaccaggcc tgggctaacc gctcgagtct agagggcccg tttaaacccg
     2941 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3001 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3061 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3121 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3181 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3241 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3301 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3361 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3421 cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag
     3481 ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac
     3541 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     3601 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     3661 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     3721 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     3781 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     3841 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     3901 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     3961 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     4021 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     4081 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     4141 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     4201 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     4261 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     4321 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     4381 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     4441 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     4501 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga
     4561 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4621 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4681 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4741 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4801 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4861 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4921 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4981 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5041 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5101 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5161 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5221 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5281 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5341 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5401 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5461 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5521 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5581 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5641 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5701 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5761 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5821 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5881 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5941 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6001 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6061 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6961 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
