LOCUS       sequence-333246-        7570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the LT-Fucci(CA) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7570)
  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 7570)
  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/HaloTag7-Geminin(1/110)_P2A_HaloTag9-Cdt(1-110)Cy-
FEATURES             Location/Qualifiers
     source          1..7570
                     /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"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             926..1810
                     /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             1847..2176
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     CDS             2186..2242
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3522..3539)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3528..3752
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3885..3904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4036..4083
                     /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             4091..5113
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4091..5113
                     /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    5243..5364
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5280..5299)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5334..5353
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5413..5429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5413..5429)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5426..5448)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5437..5453
                     /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(5461..5467,5468..5485,5486..5491))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5506..5527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5644..5661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5815..6403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6574..7365,7366..7434))
                     /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     7197..7216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7435..7539)
                     /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 ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt accaccatgg gatccgaaat cggtactggc tttccattcg acccccatta
      961 tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc
     1021 tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca
     1081 tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa
     1141 accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc
     1201 cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca
     1261 ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc
     1321 tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac
     1381 caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc
     1441 gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct
     1501 gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga
     1561 gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt
     1621 cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg
     1681 cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct
     1741 gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt ctactctgga
     1801 gatttccggt tacaaggata tccatgcaca ctggcggccg ctcgagatga atcccagtat
     1861 gaagcagaaa caagaagaaa tcaaagagaa tataaagaat agttctgtcc caagaagaac
     1921 tctgaagatg attcagcctt ctgcatctgg atctcttgtt ggaagagaaa atgagctgtc
     1981 cgcaggcttg tccaaaagga aacatcggaa tgaccactta acatctacaa cttccagccc
     2041 tggggttatt gtcccagaat ctagtgaaaa taaaaatctt ggaggagtca cccaggagtc
     2101 atttgatctt atgattaaag aaaatccatc ctctcagtat tggaaggaag tggcagaaaa
     2161 acggagaaag gcgctgggca gtggagctac taacttcagc ctgctgaagc aggctggtga
     2221 cgtcgaggag aatcctggcc cagggagcga gattggcaca gggtttccct ttgaccctca
     2281 ttacgttgag gtgctgggtg agaggatgca ctatgtcgat gtgggacccc gtgatggcac
     2341 acctgtgctg tttctccatg gcaatcctac cagctcctat gtgtggagga acatcatacc
     2401 gcatgtggct ccaacgcacc gatgtatcgc accagatctg atcgggatgg gaaagagcga
     2461 caaacctgat ctgggatact tcttcgatga ccacgtgcgc ttcatggacg cctttatcga
     2521 ggctttgggg cttgaggagg tagtgctggt catccacgat tggggatctg ccctggggtt
     2581 tcattgggcg aaacgcaatc ctgagagggt taagggcata gccttcatgg agttcatacg
     2641 gcccattcca acatgggacg aatggccgga atttgctcgg gaaacctttc aagcgttccg
     2701 cactaccgat gttgggagaa agctcatcat cgaccacaac gtgttcatcg aaggaactct
     2761 ccggatgggt gtggtacgac ccttgactga agtcgagatg gaccactata gagagccgtt
     2821 tctcaaccca gtggacagag aacctctgtg gaggttcccc aacgaacttc ccattgcagg
     2881 cgaaccagcc aatatcgtcg ccctcgttga ggagtacatg gactggttgc atcagtcacc
     2941 agtacccaag ctgttgttct ggggtacacc aggagtgctg attcctcccg cagaagcagc
     3001 tcggctggct aagtctcttc ccaactgcaa agccgtcgac attggcccag gtctgaatct
     3061 gcttcaggag gataatcctg atctgattgg cagtgagatt gccagatggc tgtcaaccct
     3121 ggaaatctcc ggctactcaa tgttgccttc cctgggcgat atccatgcac actggcggcc
     3181 gctcgaaatg gagcagcgcc gcgtcaccga cttcttcgcg cgccgccgcc ccgggccccc
     3241 ccgcatcgcg ccgcccaagc tggcctgccg cacccccagc cccgccaggc ccgcactccg
     3301 cgccccggcc tccgctacca gtggcagccg caagcgcgcc cgcccgcccg ccgcccccgg
     3361 acgcgaccag gccaggccac cggcccgcgc agccgcacgg ctgtcggtgg acgaggtttc
     3421 cagccccagt acccccgagg ccccagacat cccagcctgc ccttctccgg gccagaagat
     3481 aaagaaataa tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     3541 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3601 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3661 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3721 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3781 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3841 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4021 tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac
     4081 ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
     4141 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
     4201 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta
     4261 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct
     4321 tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
     4381 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
     4441 catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
     4501 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca
     4561 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct
     4621 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga
     4681 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
     4741 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
     4801 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
     4861 atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt
     4921 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg
     4981 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
     5041 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
     5101 ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct atgaaaggtt
     5161 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5221 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     5281 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5341 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     5401 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     5461 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5521 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5581 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5641 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5701 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5761 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5821 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5881 cccgacagga ctataaaaat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5941 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6001 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6061 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6121 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6181 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6241 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6301 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6361 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6421 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6481 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6541 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6601 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6661 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6721 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6781 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6841 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6901 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6961 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7021 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7081 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7141 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7201 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7261 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7321 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7381 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7441 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7501 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7561 acctgacgtc
//
