LOCUS       sequence-355966-        6734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV overexpression of HaloTag7 and SNAP-tag fusion in the 
            endoplasmic reticulum of mammalian cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6734)
  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 6734)
  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_CalR-HaloTag7-P30-SNAP-tag-KDEL
FEATURES             Location/Qualifiers
     source          1..6734
                     /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"
     CDS             1065..1949
                     /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(2686..2703)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2692..2916
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3049..3068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3200..3247
                     /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             3255..4277
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3255..4277
                     /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    4407..4528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4444..4463)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4498..4517
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4577..4593)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4577..4593)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4590..4612)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4601..4617
                     /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(4625..4631,4632..4649,4650..4655))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4670..4691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4808..4825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4979..5567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5059..5078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5738..6529,6530..6598))
                     /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     6361..6380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6599..6703)
                     /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 taaacttacc accatgctgc tatccgtgcc gttgctgctc ggcctcctcg
     1021 gcctggccgt cgccggaggc agcggaggca gcgaattcgg atccgaaatc ggtactggct
     1081 ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg
     1141 gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt
     1201 ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg
     1261 gtatgggcaa atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca
     1321 tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg
     1381 gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat
     1441 ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga
     1501 ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat cagaacgttt
     1561 ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct gactgaagtc gagatggacc
     1621 attaccgcga gccgttcctg aatcctgttg accgcgagcc actgtggcgc ttcccaaacg
     1681 agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact
     1741 ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg caccccaggc gttctgatcc
     1801 caccggccga agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg
     1861 gcccgggtct gaatctgctg caagaagaca acccggacct gatcggcagc gagatcgcgc
     1921 gctggctgtc tactctggag atttccggta gcggccgccc tcctcctccg ccaccgccac
     1981 caccgccgcc gccgccacca ccacctcctc cgcctcctcc accaccaccg ccaccgcctc
     2041 caccacctgg cggccgctct cggtctctcg agatggataa agactgcgaa atgaaacgca
     2101 ctaccctgga cagcccgctg ggtaaactgg aactgagcgg ttgcgagcag ggcctgcacg
     2161 aaatcatttt cctgggcaaa ggtacgagcg ctgcggatgc ggtcgaagta ccggctccgg
     2221 ctgcggttct gggtggcccg gagccgctga tgcaggctac cgcatggctg aacgcatatt
     2281 ttcaccagcc agaggcgatt gaagagttcc ctgtaccggc cctgcatcac ccggtatttc
     2341 agcaggaatc ctttacccgt caggtactgt ggaaactgct gaaagtagtg aaattcggcg
     2401 aggtgatctc ctattcccat ctggcggcgc tggctggcaa cccagcggca actgcggcgg
     2461 tgaaaaccgc tctgtctggt aatccggtgc caattctgat cccgtgccac cgtgtggttc
     2521 agggcgacct ggacgtaggt ggctacgaag gcggcctggc tgtgaaagaa tggctgctgg
     2581 cgcacgaagg tcaccgtctg ggtaaaccag gcctgggcgg aggcggagga agcggcaagg
     2641 acgagctgtg ataatctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     2701 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2761 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2821 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2881 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     2941 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3001 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3061 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3121 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3181 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     3241 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     3301 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3361 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     3421 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     3481 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     3541 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     3601 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     3661 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     3721 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     3781 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     3841 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     3901 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     3961 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4021 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4081 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4141 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     4201 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     4261 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     4321 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4381 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4441 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4501 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4561 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4621 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4681 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4801 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4861 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4921 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4981 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5041 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5101 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5161 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5221 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5281 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5341 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5401 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5461 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5521 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5581 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5641 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5701 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5761 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5821 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5881 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5941 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6001 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6061 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6121 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6181 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6241 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6301 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6361 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6421 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6481 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6541 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6601 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6661 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6721 tgccacctga cgtc
//
