LOCUS       sequence-333244-        7543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the LT-Fucci(SA) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7543)
  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 7543)
  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(30-120)
FEATURES             Location/Qualifiers
     source          1..7543
                     /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"
     CDS             3188..3460
                     /codon_start=1
                     /product="degron consisting of residues 30-120 of human 
                     Cdt1 (Zielke and Edgar, 2015)"
                     /label=Cdt1 (30-120)
                     /translation="PSPARPALRAPASATSGSRKRARPPAAPGRDQARPPARRRLRLSV
                     DEVSSPSTPEAPDIPACPSPGQKIKKSTPAAGQPPHLTSAQDQDTI"
     primer_bind     complement(3495..3512)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3501..3725
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3858..3877)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4009..4056
                     /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             4064..5086
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4064..5086
                     /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    5216..5337
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5253..5272)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5307..5326
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5386..5402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5386..5402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5399..5421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5410..5426
                     /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(5434..5440,5441..5458,5459..5464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5479..5500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5617..5634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5788..6376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5868..5887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6547..7338,7339..7407))
                     /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     7170..7189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7408..7512)
                     /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 gctcgagccc agccccgcca ggcccgcact ccgcgccccg gcctccgcta ccagtggcag
     3241 ccgcaagcgc gcccgcccgc ccgccgcccc cggacgcgac caggccaggc caccggcccg
     3301 caggagactg cggctgtcgg tggacgaggt ttccagcccc agtacccccg aggccccaga
     3361 catcccagcc tgcccttctc cgggccagaa gataaagaaa tccaccccgg cagcaggtca
     3421 gccgccccac ctgacatccg cgcaggacca ggacaccatc taatctagag ggcccgttta
     3481 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3541 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3601 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3661 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3721 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3781 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3841 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3901 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3961 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     4021 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     4081 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     4141 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     4201 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     4261 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     4321 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     4381 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     4441 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     4501 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     4561 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     4621 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     4681 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     4741 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     4801 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     4861 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     4921 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     4981 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     5041 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     5101 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     5161 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     5221 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5281 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5341 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5401 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5461 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5521 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5581 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5641 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5701 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5761 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5821 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5881 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5941 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6001 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6061 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6121 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6181 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6241 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6301 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6361 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6421 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6481 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6541 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6601 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6661 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6721 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6781 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6841 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6901 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6961 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7021 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7081 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7141 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7201 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7261 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7321 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7381 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7441 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7501 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
