LOCUS       sequence-343757-        6922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of HaloTag7 (golgi) and SNAPf-tag (membrane inner 
            leaflet) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6922)
  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 6922)
  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_Beta4Gal-HaloTag7-T2A-Lyn11-SNAPf
FEATURES             Location/Qualifiers
     source          1..6922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..56)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        228..607
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        608..811
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     762..782
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    813..831
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    834..852
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     862..886
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1264..2148
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             2164..2217
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(2867..2884)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2873..3097
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3230..3249)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3381..3428
                     /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             3436..4458
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3436..4458
                     /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    4588..4709
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4625..4644)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4679..4698
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4758..4774)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4758..4774)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4771..4793)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4782..4798
                     /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(4806..4812,4813..4830,4831..4836))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4851..4872
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4989..5006)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5160..5748)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5240..5259)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5919..6710,6711..6779))
                     /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     6542..6561
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6780..6884)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
       61 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
      121 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
      181 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
      241 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      301 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      361 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      421 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      481 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      541 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      601 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      661 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      721 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      781 tgtacggtgg gaggtctata taagcagagc tctccctatc agtgatagag atctccctat
      841 cagtgataga gatcgtcgac gagctcgttt agtgaaccgt cagatcgcct ggagacgcca
      901 tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc ggactctagc
      961 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
     1021 agatatccag caaccatgag gcttcgggag ccgctcctga gcggcagcgc cgcgatgcca
     1081 ggcgcgtccc tacagcgggc ctgccgcctg ctcgtggccg tctgcgctct gcaccttggc
     1141 gtcaccctcg tttactacct ggctggccgc gacctgagcc gcctgcccca actggtcgga
     1201 gtctccacac cgctgcagtc gacggtaccg cgggcccggg atccaccggt cgccaccgga
     1261 tccgagatcg gaaccggctt tcctttcgac cctcactacg tggaagtgct gggcgagaga
     1321 atgcactatg tggacgtggg ccccagagat ggaacccctg tgctgtttct gcacggcaac
     1381 cctaccagca gctacgtgtg gcggaacatc atccctcacg tggcccctac acacagatgt
     1441 atcgcccctg acctgatcgg catgggcaag agcgataagc ccgacctggg ctacttcttc
     1501 gacgaccacg tgcggttcat ggacgccttt attgaggccc tcggcctgga agaggtggtg
     1561 ctggttattc acgattgggg cagcgccctg ggattccact gggccaagag aaaccccgag
     1621 agagtgaagg gaatcgcctt catggaattc atcaggccca ttcctacctg ggacgagtgg
     1681 cctgagttcg ccagagagac attccaggcc ttcagaacca ccgacgtggg cagaaagctg
     1741 atcatcgacc agaacgtgtt catcgagggc accctgccta tgggagtcgt tagacctctg
     1801 accgaggtgg aaatggacca ctacagagag ccctttctga accccgtgga ccgggaacct
     1861 ctttggagat tccccaacga gctgcccatt gccggcgagc ctgctaatat tgtggccctg
     1921 gtggaagagt acatggactg gctgcatcag agccccgtgc ctaagctgct gttttgggga
     1981 acacccggcg tgctgattcc tcctgctgaa gctgccagac tggccaagag cctgcctaat
     2041 tgcaaggccg tggatatcgg ccctggcctg aatctgctgc aagaggacaa ccccgatctg
     2101 atcggctccg agattgccag atggctgagc accctggaaa tcagcggtgg ccgcggcagt
     2161 ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg
     2221 ggatgtataa aatcaaaagg gaaagacagc gcgggagcag atagtgctgg tagtgctggt
     2281 gataaagact gcgaaatgaa acgcactacc ctggacagcc cgctgggtaa actggaactg
     2341 agcggttgcg agcagggcct gcaccgcatc attttcctgg gcaaaggtac gagcgctgcg
     2401 gatgcggtcg aagtaccggc tccggctgcg gttctgggtg gcccggagcc gctgatgcag
     2461 gctaccgcat ggctgaacgc atattttcac cagccagagg cgattgaaga gttccctgta
     2521 ccggccctgc atcacccggt atttcagcag gaatccttta cccgtcaggt actgtggaaa
     2581 ctgctgaaag tagtgaaatt cggcgaggtg atctcctatt cccatctggc ggcgctggct
     2641 ggcaacccag cggcaactgc ggcggtgaaa accgctctgt ctggtaatcc ggtgccaatt
     2701 ctgatcccgt gccaccgtgt ggttcagggc gacctggacg taggtggcta cgaaggcggc
     2761 ctggctgtga aagaatggct gctggcgcac gaaggtcacc gtctgggtaa accaggcctg
     2821 ggctaaccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     2881 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2941 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3001 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3061 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3121 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3181 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3241 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3301 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3361 ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata
     3421 ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc
     3481 gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct
     3541 ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt
     3601 ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa
     3661 gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag
     3721 ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg
     3781 gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc
     3841 ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat
     3901 ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag
     3961 gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac
     4021 gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac
     4081 tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg
     4141 ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt
     4201 gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag
     4261 agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc
     4321 gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc
     4381 tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt
     4441 ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa
     4501 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4561 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4621 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4681 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4741 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4801 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4861 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4921 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4981 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5041 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5101 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5161 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5221 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5281 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5341 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5401 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5461 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5521 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5581 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5641 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5701 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5761 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5821 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5881 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5941 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6001 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6061 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6121 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6181 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6241 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6301 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6361 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6421 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6481 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6541 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6601 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6661 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6721 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6781 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6841 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6901 gtgccacctg acgtcgacgg at
//
