LOCUS       sequence-491710-        6948 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Expresses photoactivatable H2B-Cherry under EF1 promoter and can be 
            integrated into FRT site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6948)
  AUTHORS   Nozaki T, Imai R, Tanbo M, Nagashima R, Tamura S, Tani T, Joti Y, 
            Tomita M, Hibino K, Kanemaki MT, Wendt KS, Okada Y, Nagai T, 
            Maeshima K
  TITLE     Dynamic Organization of Chromatin Domains Revealed by 
            Super-Resolution Live-Cell Imaging.
  JOURNAL   Mol Cell. 2017 Jul 20;67(2):282-293.e7. doi: 
            10.1016/j.molcel.2017.06.018. Epub 2017 Jul 14.
  PUBMED    28712725
REFERENCE   2  (bases 1 to 6948)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2017.06.018"; journalName: "Mol Cell"; date: 
            "2017-07-20- 20"; volume: "67"; issue: "2"; pages: "282-293.e7"
COMMENT     Sequence Label: pEF1-alpha-H2B-PAmCherry-FRT
FEATURES             Location/Qualifiers
     source          1..6948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             45..422
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      435..444
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             441..1151
                     /codon_start=1
                     /product="photoactivatable monomeric derivative of DsRed 
                     fluorescent protein"
                     /label=PAmCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFTWDILSPQFMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMKF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALK
                     GEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(586..604)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     854..873
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1218..1259
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1297..1314)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1303..1527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1660..1679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1811..1858
                     /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             1866..2888
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1866..2888
                     /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    3018..3139
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3055..3074)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3109..3128
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3188..3204)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3188..3204)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3201..3223)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3212..3228
                     /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(3236..3242,3243..3260,3261..3266))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3281..3302
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3419..3436)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3590..4178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3670..3689)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4349..5140,5141..5209))
                     /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     4972..4991
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5210..5314)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5389..5408)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5696..6874
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5927..6865
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6822..6842
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     6891..6910
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6891..6909
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gatccactag tccagtgtgg tggaattctg cagatctagc tagcatgcca gagccagcga
       61 agtctgctcc cgccccgaaa aagggctcca agaaggcggt gactaaggcg cagaagaaag
      121 gcggcaagaa gcgcaagcgc agccgcaagg agagctattc catctatgtg tacaaggttc
      181 tgaagcaggt ccaccctgac accggcattt cgtccaaggc catgggcatc atgaattcgt
      241 ttgtgaacga cattttcgag cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca
      301 agcgctcgac catcacctcc agggagatcc agacggccgt gcgcctgctg ctgcctgggg
      361 agttggccaa gcacgccgtg tccgagggta ctaaggccat caccaagtac accagcgcta
      421 aggatccacc ggtcgccacc atggtgagca agggcgagga ggataacatg gccatcatta
      481 aggagttcat gcgcttcaag gtgcacatgg aggggtccgt gaacggccac gtgttcgaga
      541 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
      601 ccaagggtgg ccccctgccc ttcacctggg acatcctgtc ccctcaattc atgtacggct
      661 ccaatgccta cgtgaagcac cccgccgaca tccccgacta ctttaagctg tccttccccg
      721 agggcttcaa gtgggagcgc gtgatgaaat tcgaggacgg cggcgtggtg accgtgaccc
      781 aggactcctc cctgcaggac ggtgagttca tctacaaggt gaagctgcgc ggcaccaact
      841 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc ctctccgagc
      901 ggatgtaccc cgaggacggc gccctgaagg gcgaggtcaa gccgagagtg aagctgaagg
      961 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     1021 tgcccggcgc ctacaacgtc aaccgcaagt tggacatcac ctcacacaac gaggactaca
     1081 ccatcgtgga acagtacgaa cgtgccgagg gccgccactc caccggcggc atggacgagc
     1141 tgtacaagta agcggccgct tttttcctta tccagcacag tggcggccgc tcgagtctag
     1201 agggcccgcg gttcgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc
     1261 gtaccggtta gtaatgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     1321 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     1381 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     1441 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     1501 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     1561 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1621 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1681 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1741 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1801 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct
     1861 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     1921 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     1981 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     2041 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     2101 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     2161 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     2221 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     2281 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     2341 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     2401 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     2461 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     2521 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     2581 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     2641 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     2701 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     2761 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     2821 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     2881 aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     2941 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3001 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3061 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3121 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     3181 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3241 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3301 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3361 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3421 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3481 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3541 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3601 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3661 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3721 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3781 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3841 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3901 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3961 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4021 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4081 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4141 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4201 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4261 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4321 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4381 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4441 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4501 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4561 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4621 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4681 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4741 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4801 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4861 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4921 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4981 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5041 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5101 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5161 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5221 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5281 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5341 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     5401 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     5461 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     5521 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     5581 acattgatta ttgactaggc ttttgcaaaa agctttgcaa agatggataa agttttaaac
     5641 agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaattggctc
     5701 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     5761 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     5821 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     5881 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
     5941 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
     6001 cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag
     6061 ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg
     6121 gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga
     6181 taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga
     6241 tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg
     6301 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
     6361 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
     6421 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
     6481 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
     6541 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
     6601 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
     6661 ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
     6721 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
     6781 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
     6841 tcaaagtttt tttcttccat ttcaggtgtc gtgaggaatt agcttggtac taatacgact
     6901 cactataggg agacccaagc tggctaggta agcttggtac cgagctcg
//
