LOCUS       sequence-308113-        8142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector for expressing chimeric human histone H2B fused 
            with the ""Medium"" variant of the monomeric color-changing 
            fluorescent timer (FT) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8142)
  AUTHORS   Eastman AE, Chen X, Hu X, Hartman AA, Pearlman Morales AM, Yang C, 
            Lu J, Kueh HY, Guo S
  TITLE     Resolving Cell Cycle Speed in One Snapshot with a Live-Cell 
            Fluorescent Reporter.
  JOURNAL   Cell Rep. 2020 Jun 23;31(12):107804. doi: 
            10.1016/j.celrep.2020.107804.
  PUBMED    32579930
REFERENCE   2  (bases 1 to 8142)
  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: "Cell Rep.";
            date: "2020-06-23"; volume: "31(12):107804. doi"; pages: " 
            10.1016/j.celrep.2020.107804"
COMMENT     Sequence Label: pSCMV-H2B-FT-Medium
FEATURES             Location/Qualifiers
     source          1..8142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(202..221)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(349..371)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(565..584)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     684..706
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(744..762)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        830..934
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(935..1003,1004..1795)
                     /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     complement(1153..1172)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1966..2554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2455..2474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2708..2725
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2842..2863
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2878..2883,2884..2901,2902..2908)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2916..2932
                     /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)."
     CDS             complement(3518..4117)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3601..3621)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4098..4117
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4142..4641)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4190..4209)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4583..4606
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     enhancer        4677..5056
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5057..5260
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5211..5231
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5305..5324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5305..5323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5343..5720
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     CDS             5727..6437
                     /codon_start=1
                     /product="blue-to-red fluorescent timer derivative of 
                     mCherry (Subach et al., 2009)"
                     /label=Medium-FT
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHLEGSVDGHEFEIEGEGEGRPYEG
                     TQSAKLKVTKGGPLPFAWDILSPQFMYGSRAYVKHPADIPDYWKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERIYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVLLPGAYNVNIKMDITSHNEDYTIVEQCERA
                     EGHHSTGGMDELYK"
     primer_bind     complement(5872..5890)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6140..6159
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(6465..6483)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6466..6483)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6503..6520)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6509..6716
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(6740..7156)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(6756..6772)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(6788..6810)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    7223..7564
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             join(7628..8142,1..2)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 ttcccccctt tttctggaga ctaaataaaa tcttttattt tatctatggc tcgtactcta
       61 taggcttcag ctggtgatat tgttgagtca aaactagagc ctggaccact gatatcctgt
      121 ctttaacaaa ttggactaat cgcctctacg ccggacgcat cgtggccggc atcaccggcg
      181 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
      241 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
      301 ggggactgtt gggcgccatc tccttgcatg caccattcct tgcgccgtcg ttttacaacg
      361 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
      421 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
      481 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
      541 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
      601 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
      661 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
      721 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
      781 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      841 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      901 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
      961 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1021 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1081 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1141 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     1201 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1261 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1321 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1381 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1441 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     1501 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1561 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1621 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     1681 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     1741 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     1801 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     1861 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     1921 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1981 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2041 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2101 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2161 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2221 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2281 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2341 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2401 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2461 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2521 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2581 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2641 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2701 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     2761 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2821 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     2881 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacaa
     2941 ttctccccag ctctttctgc gctgtccata atgaactatt tcaggagtat tggagtatgt
     3001 ggattcaaaa cagtggctaa ttgagtcagg attacttaat attcgactcc taccctcaga
     3061 atgttgttct ccaacttcaa gaaactgtta cccatgaaag acccccgctg acgggtagtc
     3121 aatcactcag aggagaccct cccaaggaac agcgagacca caagtcggat gcaactgcaa
     3181 gagggtttat tggatacacg ggtacccggg cgacgcagtc tatcggagga ctggcgcgcc
     3241 gagtgagggg ttgtgggctc ttttattgag ctcggggagc agaagcgcgc gaacagaagc
     3301 gagaagcgaa ctgattggtt agttcaaata aggcacaggg tcatttcagg tccttggggc
     3361 accctggaaa catctgatgg ttctctagct tgccaaacct acaggtgggg tctttcattc
     3421 cccccttttt ctggagacta aataaaatct tttattttat cgatgcatgg ggtcgtgcgc
     3481 tcctttcggt cgggcgctgc gggtcgtggg gcgggcgtca ggcaccgggc ttgcgggtca
     3541 tgcaccaggt gcgcggtcct tcgggcacct cgacgtcggc ggtgacggtg aagccgagcc
     3601 gctcgtagaa ggggaggttg cggggcgcgg aggtctccag gaaggcgggc accccggcgc
     3661 gctcggccgc ctccactccg gggagcacga cggcgctgcc cagacccttg ccctggtggt
     3721 cgggcgagac gccgacggtg gccaggaacc acgcgggctc cttgggccgg tgcggcgcca
     3781 ggaggccttc catctgttgc tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg
     3841 ggccgatctc ggcgaacacc gcccccgctt cgacgctctc cggcgtggtc cagaccgcca
     3901 ccgcggcgcc gtcgtccgcg acccacacct tgccgatgtc gagcccgacg cgcgtgagga
     3961 agagttcttg cagctcggtg acccgctcga tgtggcggtc cggatcgacg gtgtggcgcg
     4021 tggcggggta gtcggcgaac gcggcggcga gggtgcgtac ggccctgggg acgtcgtcgc
     4081 gggtggcgag gcgcaccgtg ggcttgtact cggtcatggt aagctagctt gggctgcagg
     4141 tcgaaaggcc cggagatgag gaagaggaga acagcgcggc agacgtgcgc ttttgaagcg
     4201 tgcagaatgc cgggcctccg gaggaccttc gggcgcccgc cccgcccctg agcccgcccc
     4261 tgagcccgcc cccggaccca ccccttccca gcctctgagc ccagaaagcg aaggagcaaa
     4321 gctgctattg gccgctgccc caaaggccta cccgcttcca ttgctcagcg gtgctgtcca
     4381 tctgcacgag actagtgaga cgtgctactt ccatttgtca cgtcctgcac gacgcgagct
     4441 gcggggcggg ggggaacttc ctgactaggg gaggagtaga aggtggcgcg aaggggccac
     4501 caaagaacgg agccggttgg cgcctaccgg tggatgtgga atgtgtgcga ggccagaggc
     4561 cacttgtgta gcgccaagtg cccagcgggg ctgctaaagc gcatgctcca gactgccttg
     4621 ggaaaagcgc ctcccctacc cggtagaatt cgatgtacgg gccagatata cgcgttgaca
     4681 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     4741 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     4801 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     4861 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     4921 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     4981 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     5041 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     5101 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     5161 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     5221 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
     5281 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttggca
     5341 ccatgccaga gccagcgaag tctgctcccg ccccgaaaaa gggctccaag aaggcggtga
     5401 ctaaggcgca gaagaaagac ggcaagaagc gcaagcgcag ccgcaaggag agctattcca
     5461 tctatgtgta caaggttctg aagcaggtcc accctgacac cggcatttcg tccaaggcca
     5521 tgggcatcat gaattcgttt gtgaacgaca ttttcgagcg catcgcaggt gaggcttccc
     5581 gcctggcgca ttacaacaag cgctcgacca tcacctccag ggagatccag acggccgtgc
     5641 gcctgctgct gcctggggag ttggccaagc acgccgtgtc cgagggtact aaggccgtca
     5701 ccaagtacac cagcgctaag gatccgatgg taagcaaggg cgaggaggat aacatggcca
     5761 tcatcaagga attcatgcgt ttcaaggtgc acctggaggg ctccgtggac ggccacgagt
     5821 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagagc gccaagctga
     5881 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
     5941 acggctccag ggcctacgtg aagcaccccg ccgacatccc cgactactgg aagctgtcct
     6001 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggatggcggc gtggtgaccg
     6061 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     6121 ccaacttccc ttccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
     6181 ccgagcggat ataccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
     6241 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
     6301 tgctgctgcc cggcgcctac aacgtcaaca tcaagatgga catcacctcc cacaacgagg
     6361 actacaccat cgttgaacag tgcgaacgcg ccgagggcca ccattccacc ggcggcatgg
     6421 acgagctgta caagtaactc gagcatgcat ctagagggcc ctattctata gtgtcaccta
     6481 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     6541 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     6601 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     6661 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgtc
     6721 gagagatcga tctaattccg gcgcctagag aaggagtgag ggctggataa agggaggatc
     6781 gaggcggggt cgaacgagga ggttcaaggg ggagagacgg ggcggatgga ggaagaggag
     6841 gcggaggctt agggtgtaca aagggcttga cccagggagg ggggtcaaaa gccaaggctt
     6901 cccaggtcac gatgtagggg acctggtctg ggtgtccatg cgggccaggt gaaaagacct
     6961 tgatcttaac ctgggtgatg aggtctcggt taaaggtgcc gtctcgcggc catccgacgt
     7021 taaaggttgg ccattctgca gagcagaagg taacccaacg tctcttcttg acatctaccg
     7081 actggttgtg agcgatccgc tcgacatctt tccagtgacc taaggtcaaa cttaagggag
     7141 tggtaacagt ctggccctaa ttttcagaca aatacagaaa cacagtcaga cagagacaac
     7201 acagaacgat gctgcagcgc tgcagcagac aagacgcgcg gcttcggttc caaaccgaaa
     7261 gcaaaaattc agacggaggc gggaactgtt ttaggttctc gtctcctacc agaaccacat
     7321 atcctgacgg ggtcggattc cacatcgact cccttcctca ggtcgggcca caaaaacggc
     7381 ccccaaagtc cctgggacgt ctcccagggt tgcggccggg tgttcagaac tcgtcagttc
     7441 caccacgggt ccgccagata cagagctagt tagctaacta gtacagacgc aggcgcaaac
     7501 attagatgcc ggcacaaaca cgcacaaaga cagagacaga cacgaaacga ccgctggcca
     7561 gcttacctcc cggcgggggg gtcggtggtc cctgggcagg ggtctccaaa tctcggtgga
     7621 acctccaaat gaaagacccc cgaggtgggc agtcaatcaa tctgaggaga ccctcccaag
     7681 gatcagcgag tccacgattc ggatgcaaac agcaagaggc tttattggga atacgggtac
     7741 ccgggcgacg cagtctatcg gaggactggc gcgccgagtg aggggttgtg ggctctttta
     7801 ttgagctcgg ggagcagaag cgcgcgaaca gaagcgagaa gcgaactgat tggttagttc
     7861 aaataaggca cagggtcatt tcaggtcctt ggggcaccct ggaaacatct gatggttctc
     7921 tagaaactgc tgagggcggg accgcatctg gggaccatct gttcttggcc ctgagccggg
     7981 gcaggaactg cttaccacag atatcctgtt tggcccatat tctgctgtct ctctgttcct
     8041 aaccttgatc tgaacttctc tattctcagt tatgtatttt ccatgccttg caaaatggcg
     8101 ttacttaagc tagcttgcca aacctacagg tggggtcttt ca
//
