LOCUS       sequence-347596-        7345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lyzosomal expression of HaloTag7 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7345)
  AUTHORS   Frei MS, Tarnawski M, Roberti MJ, Koch B, Hiblot J, Johnsson K
  TITLE     Engineered HaloTag variants for fluorescence lifetime multiplexing.
  JOURNAL   Nat Methods. 2021 Dec 16. pii: 10.1038/s41592-021-01341-x. doi: 
            10.1038/s41592-021-01341-x.
  PUBMED    34916672
REFERENCE   2  (bases 1 to 7345)
  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 
            Methods. 2021 Dec 16. pii: 10.1038/s41592-021-01341-x. doi: 
            10.1038/s41592-021-01341-x."
COMMENT     Sequence Label: pcDNA5/FRT/TO_LAMP1_HaloTag7
FEATURES             Location/Qualifiers
     source          1..7345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(18..122)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(197..216)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        388..767
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        768..971
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     922..942
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    973..991
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    994..1012
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1022..1046
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1194..1203
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1196..1279,1280..2446)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     CDS             2522..3406
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(3450..3467)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3456..3680
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3813..3832)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3964..4011
                     /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             4019..5041
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4019..5041
                     /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    5171..5292
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5208..5227)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5262..5281
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5341..5357)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5341..5357)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5354..5376)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5365..5381
                     /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(5389..5395,5396..5413,5414..5419))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5434..5455
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5572..5589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5743..6331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5823..5842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6502..7293,7294..7345,1..17))
                     /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     7125..7144
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
       61 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
      121 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
      181 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
      241 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
      301 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      361 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      421 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      481 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      541 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      601 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      661 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      721 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      781 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      841 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      901 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      961 taagcagagc tctccctatc agtgatagag atctccctat cagtgataga gatcgtcgac
     1021 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
     1081 atagaagaca ccgggaccga tccagcctcc ggactctagc gtttaaactt aagcttggta
     1141 ccgagctcgg atccactagt ccagtgtggt ggaattctgc agatatccag caaccatggc
     1201 ggcccccggc agcgcccggc gacccctgct gctgctactg ctgttgctgc tgctcggcct
     1261 catgcattgt gcgtcagcag caatgtttat ggtgaaaaat ggcaacggga ccgcgtgcat
     1321 aatggccaac ttctctgctg ccttctcagt gaactacgac accaagagtg gccctaagaa
     1381 catgaccttt gacctgccat cagatgccac agtggtgctc aaccgcagct cctgtggaaa
     1441 agagaacact tctgacccca gtctcgtgat tgcttttgga agaggacata cactcactct
     1501 caatttcacg agaaatgcaa cacgttacag cgtccagctc atgagttttg tttataactt
     1561 gtcagacaca caccttttcc ccaatgcgag ctccaaagaa atcaagactg tggaatctat
     1621 aactgacatc agggcagata tagataaaaa atacagatgt gttagtggca cccaggtcca
     1681 catgaacaac gtgaccgtaa cgctccatga tgccaccatc caggcgtacc tttccaacag
     1741 cagcttcagc aggggagaga cacgctgtga acaagacagg ccttccccaa ccacagcgcc
     1801 ccctgcgcca cccagcccct cgccctcacc cgtgcccaag agcccctctg tggacaagta
     1861 caacgtgagc ggcaccaacg ggacctgcct gctggccagc atggggctgc agctgaacct
     1921 cacctatgag aggaaggaca acacgacggt gacaaggctt ctcaacatca accccaacaa
     1981 gacctcggcc agcgggagct gcggcgccca cctggtgact ctggagctgc acagcgaggg
     2041 caccaccgtc ctgctcttcc agttcgggat gaatgcaagt tctagccggt ttttcctaca
     2101 aggaatccag ttgaatacaa ttcttcctga cgccagagac cccgccttta aagctgccaa
     2161 cggctccctg cgagcgctgc aggccacagt cggcaattcc tacaagtgca acgcggagga
     2221 gcacgtccgt gtcacgaagg cgttttcagt caatatattc aaagtgtggg tccaggcttt
     2281 caaggtggaa ggtggccagt ttggctctgt ggaggagtgt ctgctggacg agaacagcat
     2341 gctgatcccc atcgctgtgg gtggtgccct ggcggggctg gtcctcatcg tcctcatcgc
     2401 ctacctcgtc ggcaggaaga ggagtcacgc aggctaccag actatcggct ccaccggctc
     2461 cacaggctcc accggcgcgg tcgacggtac cgcgggcccg ggatccatcg ccaccggatc
     2521 cgaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat
     2581 gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc
     2641 gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat
     2701 tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga
     2761 cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct
     2821 ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg
     2881 cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc
     2941 agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat
     3001 catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac
     3061 tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc gcgagccact
     3121 gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt
     3181 cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt tctggggcac
     3241 cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg
     3301 caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc cggacctgat
     3361 cggcagcgag atcgcgcgct ggctgtctac tctggagatt tccggttaac cgctcgagtc
     3421 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3481 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3541 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3601 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3661 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3721 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3781 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3841 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3901 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     3961 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     4021 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     4081 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     4141 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     4201 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     4261 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     4321 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     4381 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     4441 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     4501 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     4561 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     4621 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     4681 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     4741 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     4801 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     4861 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     4921 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     4981 agaagtactt gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     5041 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5101 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5161 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5221 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5281 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5341 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5401 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5461 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5521 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5581 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5641 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5701 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5761 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5821 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5881 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5941 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6001 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6061 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6121 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6181 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6241 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6301 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6361 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6421 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6481 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6541 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6601 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6661 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6721 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6781 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6841 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6901 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6961 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7021 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7081 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7141 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7201 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7261 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7321 aaaaaaggga ataagggcga cacgg
//
