LOCUS       sequence-343743-        7345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lyzosomal expression of HaloTag9 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_HaloTag9
FEATURES             Location/Qualifiers
     source          1..7345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(131..150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        322..701
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        702..905
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     856..876
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    907..925
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    928..946
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     956..980
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1128..1137
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1130..1213,1214..2380)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     CDS             2456..3340
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDH
                     NVFIEGTLRMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(3384..3401)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3390..3614
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3747..3766)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3898..3945
                     /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             3953..4975
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3953..4975
                     /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    5105..5226
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5142..5161)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5196..5215
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5275..5291)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5275..5291)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5288..5310)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5299..5315
                     /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(5323..5329,5330..5347,5348..5353))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5368..5389
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5506..5523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5677..6265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5757..5776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6436..7227,7228..7296))
                     /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     7059..7078
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7297..7345,1..56))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
       61 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
      121 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
      181 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      241 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      301 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      361 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      421 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      481 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      541 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      601 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      661 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      721 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      781 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      841 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      901 gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt cgacgagctc
      961 gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
     1021 gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt ggtaccgagc
     1081 tcggatccac tagtccagtg tggtggaatt ctgcagatat ccagcaacca tggcggcccc
     1141 cggcagcgcc cggcgacccc tgctgctgct actgctgttg ctgctgctcg gcctcatgca
     1201 ttgtgcgtca gcagcaatgt ttatggtgaa aaatggcaac gggaccgcgt gcataatggc
     1261 caacttctct gctgccttct cagtgaacta cgacaccaag agtggcccta agaacatgac
     1321 ctttgacctg ccatcagatg ccacagtggt gctcaaccgc agctcctgtg gaaaagagaa
     1381 cacttctgac cccagtctcg tgattgcttt tggaagagga catacactca ctctcaattt
     1441 cacgagaaat gcaacacgtt acagcgtcca gctcatgagt tttgtttata acttgtcaga
     1501 cacacacctt ttccccaatg cgagctccaa agaaatcaag actgtggaat ctataactga
     1561 catcagggca gatatagata aaaaatacag atgtgttagt ggcacccagg tccacatgaa
     1621 caacgtgacc gtaacgctcc atgatgccac catccaggcg tacctttcca acagcagctt
     1681 cagcagggga gagacacgct gtgaacaaga caggccttcc ccaaccacag cgccccctgc
     1741 gccacccagc ccctcgccct cacccgtgcc caagagcccc tctgtggaca agtacaacgt
     1801 gagcggcacc aacgggacct gcctgctggc cagcatgggg ctgcagctga acctcaccta
     1861 tgagaggaag gacaacacga cggtgacaag gcttctcaac atcaacccca acaagacctc
     1921 ggccagcggg agctgcggcg cccacctggt gactctggag ctgcacagcg agggcaccac
     1981 cgtcctgctc ttccagttcg ggatgaatgc aagttctagc cggtttttcc tacaaggaat
     2041 ccagttgaat acaattcttc ctgacgccag agaccccgcc tttaaagctg ccaacggctc
     2101 cctgcgagcg ctgcaggcca cagtcggcaa ttcctacaag tgcaacgcgg aggagcacgt
     2161 ccgtgtcacg aaggcgtttt cagtcaatat attcaaagtg tgggtccagg ctttcaaggt
     2221 ggaaggtggc cagtttggct ctgtggagga gtgtctgctg gacgagaaca gcatgctgat
     2281 ccccatcgct gtgggtggtg ccctggcggg gctggtcctc atcgtcctca tcgcctacct
     2341 cgtcggcagg aagaggagtc acgcaggcta ccagactatc ggctccaccg gctccacagg
     2401 ctccaccggc gcggtcgacg gtaccgcggg cccgggatcc atcgccaccg gatccgaaat
     2461 cggtactggc tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta
     2521 cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc
     2581 ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc
     2641 agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca
     2701 cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat
     2761 tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa
     2821 aggtattgca tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt
     2881 tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga
     2941 tcataacgtt tttatcgagg gtacgctgcg tatgggtgtc gtccgcccgc tgactgaagt
     3001 cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg
     3061 cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga
     3121 atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg
     3181 cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc
     3241 tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag
     3301 cgagatcgcg cgctggctgt ctactctgga gatttccggt taaccgctcg agtctagagg
     3361 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3421 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3481 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3541 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3601 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3661 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3721 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3781 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3841 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     3901 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     3961 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     4021 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     4081 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     4141 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     4201 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     4261 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     4321 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     4381 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     4441 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     4501 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     4561 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     4621 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     4681 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     4741 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     4801 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     4861 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     4921 acttgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     4981 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     5041 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5101 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5161 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5221 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5281 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5341 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5401 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5461 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5521 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5581 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5641 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5701 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5761 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5821 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5881 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5941 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6001 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6061 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6121 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6181 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6241 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6301 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6361 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6421 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6481 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6541 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6601 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6661 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6721 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6781 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6841 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6901 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6961 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7021 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7081 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7141 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7201 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7261 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7321 aagcatttat cagggttatt gtctc
//
