LOCUS       sequence-491711-        7151 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Expresses H2B-Halo 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 7151)
  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 7151)
  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-HaloTag-FRT
FEATURES             Location/Qualifiers
     source          1..7151
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(21..905)
                     /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             complement(972..1349)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(1441..1460)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1442..1460)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1477..2655)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1486..2424
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(1509..1529)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     2943..2962
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3037..3141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3142..3210,3211..4002)
                     /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(3360..3379)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4173..4761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4662..4681
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4915..4932
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5049..5070
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5085..5090,5091..5108,5109..5115)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5123..5139
                     /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)."
     primer_bind     5128..5150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5147..5163
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5147..5163
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5212..5333
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5223..5242)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5277..5296
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5463..6485)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(5463..6485)
                     /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
                     "
     protein_bind    complement(6493..6540)
                     /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)."
     primer_bind     6672..6691
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6824..7048
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7037..7054
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(7092..7133)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
ORIGIN
        1 tagactcgag cggccgctta accggaaatc tccagagtag acagccagcg cgcgatctcg
       61 ctgccgatca ggtccgggtt gtcttcttgc agcagattca gacccgggcc gatgtccaca
      121 gccttgcagt taggcaggct tttggccagg cgagcggctt cggccggtgg gatcagaacg
      181 cctggggtgc cccagaacag cagcttcggg acaggggact ggtgcagcca gtccatgtat
      241 tcttcgacca gcgcgacgat gttcgctggc tcaccggcga ttggcagctc gtttgggaag
      301 cgccacagtg gctcgcggtc aacaggattc aggaacggct cgcggtaatg gtccatctcg
      361 acttcagtca gcgggcggac gacacccatc ggcagcgtac cctcgataaa aacgttctga
      421 tcgatgatca gcttgcggcc gacgtcggtg gtgcggaagg cctggaaggt ctcgcgggca
      481 aattctggcc attcgtccca ggtcgggata gggcggatga actccataaa tgcaatacct
      541 ttgacgcgct ctggattgcg cttggcccag tggaaaccca gagcggagcc ccagtcgtga
      601 atgaccagga cgacctcttc cagacccagg gcttcgatga aggcatccat gaagcggacg
      661 tggtcgtcga agaaataacc caggtctggt ttgtcggatt tgcccatacc gatcaggtct
      721 ggagcaatgc agcgatgggt cggtgcaaca tgcgggatga tgttgcgcca cacgtaggag
      781 gaggtcgggt taccgtgcag gaacagcaca ggggtgccat cgcgcggacc aacatcgacg
      841 tagtgcatgc gctcgcccag gacttccaca taatgggggt cgaatggaaa gccagtaccg
      901 atttcggatc cagacccgcc acctcctgag cctccaccgc cacttagcgg ccgccactgt
      961 gctggatgat ccttagcgct ggtgtacttg gtgatggcct tagtaccctc ggacacggcg
     1021 tgcttggcca actccccagg cagcagcagg cgcacggccg tctggatctc cctggaggtg
     1081 atggtcgagc gcttgttgta atgcgccagg cgggaagcct cacctgcgat gcgctcgaaa
     1141 atgtcgttca caaacgaatt catgatgccc atggccttgg acgaaatgcc ggtgtcaggg
     1201 tggacctgct tcagaacctt gtacacatag atggaatagc tctccttgcg gctgcgcttg
     1261 cgcttcttgc cgcctttctt ctgcgcctta gtcaccgcct tcttggagcc ctttttcggg
     1321 gcgggagcag acttcgctgg ctctggcatg gtggcggtac cgtcgacatc tgcagaattc
     1381 caccacactg gactagtgga tccgagctcg gtaccaagct tacctagcca gcttgggtct
     1441 ccctatagtg agtcgtatta gtaccaagct aattcctcac gacacctgaa atggaagaaa
     1501 aaaactttga accactgtct gaggcttgag aatgaaccaa gatccaaact caaaaagggc
     1561 aaattccaag gagaattaca tcaagtgcca agctggccta acttcagtct ccacccactc
     1621 agtgtgggga aactccatcg cataaaaccc ctccccccaa cctaaagacg acgtactcca
     1681 aaagctcgag aactaatcga ggtgcctgga cggcgcccgg tactccgtgg agtcacatga
     1741 agcgacggct gaggacggaa aggccctttt cctttgtgtg ggtgactcac ccgcccgctc
     1801 tcccgagcgc cgcgtcctcc attttgagct ccctgcagca gggccgggaa gcggccatct
     1861 ttccgctcac gcaactggtg ccgaccgggc cagccttgcc gcccagggcg gggcgataca
     1921 cggcggcgcg aggccaggca ccagagcagg ccggccagct tgagactacc cccgtccgat
     1981 tctcggtggc cgcgctcgca ggccccgcct cgccgaacat gtgcgctggg acgcacgggc
     2041 cccgtcgccg cccgcggccc caaaaaccga aataccagtg tgcagatctt ggcccgcatt
     2101 tacaagacta tcttgccaga aaaaaagcgt cgcagcaggt catcaaaaat tttaaatggc
     2161 tagagactta tcgaaagcag cgagacaggc gcgaaggtgc caccagattc gcacgcggcg
     2221 gccccagcgc ccaggccagg cctcaactca agcacgaggc gaaggggctc cttaagcgca
     2281 aggcctcgaa ctctcccacc cacttccaac ccgaagctcg ggatcaagaa tcacgtactg
     2341 cagccaggtg gaagtaattc aaggcacgca agggccataa cccgtaaaga ggccaggccc
     2401 gcgggaacca cacacggcac ttacctgtgt tctggcggca aacccgttgc gaaaaagaac
     2461 gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag
     2521 ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg caccggttca
     2581 attgccgacc cctcccccca acttctcggg gactgtgggc gatgtgcgct ctgcccactg
     2641 acgggcaccg gagccaattc ccactccttt caagacctag aaggtccatt agctgcaaag
     2701 attcctctct gtttaaaact ttatccatct ttgcaaagct ttttgcaaaa gcctagtcaa
     2761 taatcaatgt caacgcgtat atctggcccg tacatcgcga agcagcgcaa aacgcctaac
     2821 cctaagcaga ttcttcatgc aattgtcggt caagccttgc cttgttgtag cttaaatttt
     2881 gctcgcgcac tactcagcga cctccaacac acaagcaggg agcagatact ggcttaacta
     2941 tgcggcatca gagcagattg tactgagagt gcaccatagg ggatcgggag atctcccgat
     3001 ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3061 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3121 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3181 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3241 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3301 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3361 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3421 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3481 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3541 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3601 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3661 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3721 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3781 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3841 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3901 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3961 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     4021 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4081 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4141 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4201 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4261 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4321 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4381 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4441 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4501 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4561 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4621 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4681 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4741 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4801 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4861 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4921 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     4981 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5041 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5101 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5161 gaccatgatt acgccaagct ctagctagag gtcgacggta tacagacatg ataagataca
     5221 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     5281 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttggggtgg
     5341 gcgaagaact ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa
     5401 acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtagtacg
     5461 tgctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact
     5521 tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca
     5581 gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg
     5641 aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc
     5701 cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc
     5761 tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc
     5821 ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca
     5881 ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa
     5941 agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt
     6001 tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat
     6061 tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca
     6121 gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc
     6181 aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgctg
     6241 aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa
     6301 acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc
     6361 cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg
     6421 gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc
     6481 tttttggcca aggaagttcc tatactttct agagaatagg aacttcggaa taggaacttc
     6541 taggtacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     6601 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     6661 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6721 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg
     6781 ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca
     6841 tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca
     6901 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
     6961 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
     7021 acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta aactcattac
     7081 taaccggtac gcgtagaatc gagaccgagg agagggttag ggataggctt accttcgaac
     7141 cgcgggccct c
//
