LOCUS       sequence-492512-        8311 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Expresses H2B-Halo under CAG promoter and can be randomly integrated
            by PiggyBac transposase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8311)
  AUTHORS   Nagashima R, Hibino K, Ashwin SS, Babokhov M, Fujishiro S, Imai R, 
            Nozaki T, Tamura S, Tani T, Kimura H, Shribak M, Kanemaki MT, Sasai 
            M, Maeshima K
  TITLE     Single nucleosome imaging reveals loose genome chromatin networks 
            via active RNA polymerase II.
  JOURNAL   J Cell Biol. 2019 Mar 1. pii: jcb.201811090. doi: 
            10.1083/jcb.201811090.
  PUBMED    30824489
REFERENCE   2  (bases 1 to 8311)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol. 2019 Mar 1. pii: jcb.201811090. doi: 10.1083/jcb.201811090."
COMMENT     Sequence Label: pPB-CAG-IB-H2B-HaloTag
FEATURES             Location/Qualifiers
     source          1..8311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(180..456)
                     /label=chicken beta-actin promoter
     enhancer        458..837
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   1123..1157
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1333..1352)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1334..1352)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1359..1376)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1359..1375)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1368..1390)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1517..1972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1604..1623)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1814..1835
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1998..2102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2103..2171,2172..2963)
                     /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(2321..2340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3134..3722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3623..3642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3876..3893
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4010..4031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4046..4051,4052..4069,4070..4076)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4084..4100
                     /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     4089..4111
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4108..4124
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4108..4124
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4145..4163
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   4293..4355
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     misc_feature    join(5470..5478,5479..5481,5482..6056)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5643..5662)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     5872..5889
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(6081..6965)
                     /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(7032..7409)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(7447..7466)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          join(7474..8311,1..179)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(7528..7550)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 tcacgcagcc acagaaaaga aacaagccgt cattaaacca agcgctaatt acagcccgga
       61 ggagaagggc cgtcccgccc gctcacctgt gggagtaacg cggtcagtca gagccggggc
      121 gggcggcgcg aggcggcggc ggagcggggc acggggcgaa ggcagcgcgc agcgactccc
      181 gcccgccgcg cgcttcgctt tttatagggc cgccgccgcc gccgcctcgc cataaaagga
      241 aactttcgga gcgcgccgct ctgattggct gccgccgcac ctctccgcct cgccccgccc
      301 cgcccctcgc cccgccccgc cccgcctggc gcgcgccccc cccccccccc gcccccatcg
      361 ctgcacaaaa taattaaaaa ataaataaat acaaaattgg gggtggggag gggggggaga
      421 tggggagagt gaagcagaac gtggggctca cctcgaccat ggtaatagcg atgactaata
      481 cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg
      541 cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat
      601 gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc
      661 ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg
      721 gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc
      781 tatgaactaa tgaccccgta attgattact attaataact agtcaataat caatgtcgac
      841 cgggatctga tatctataac aagaaaatat atatataata agttatcacg taagtagaac
      901 atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc
      961 atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg
     1021 acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg
     1081 gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc
     1141 agactatctt tctagggtta aaaaagattt gcgctttact cgacctaaac tttaaacacg
     1201 tcatagaatc ttcgtttgac aaaaaccaca ttgtggccaa gctgtgtgac gcgacgcgcg
     1261 ctaaagaatg gcaaaccaag tcgcgcgagg tacctctctt aaggtagcgc gatcgcacgc
     1321 gtacccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt
     1381 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     1441 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     1501 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1561 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1621 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1681 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1741 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1801 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1861 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1921 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     1981 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2041 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2101 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2161 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2221 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2281 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2341 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     2401 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2461 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     2521 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2581 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2641 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2701 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     2761 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2821 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     2881 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     2941 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3001 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3061 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3121 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3181 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3241 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3301 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3361 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3421 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3481 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3541 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3601 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3661 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3721 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3781 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3841 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3901 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3961 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     4021 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     4081 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     4141 tcgaaattaa ccctcactaa agggaacaaa agctggagct agggataaca gggtaatggc
     4201 gcgccttaat taactataac ggtcctaagg tagcgagctc ggtattcacg acagcaggct
     4261 gaataataaa aaaattagaa actattattt aaccctagaa agataatcat attgtgacgt
     4321 acgttaaaga taatcatgcg taaaattgac gcatgtgttt tatcggtctg tatatcgagg
     4381 tttatttatt aatttgaata gatattaagt tttattatat ttacacttac atactaataa
     4441 taaattcaac aaacaattta tttatgttta tttatttatt aaaaaaaaac aaaaactcaa
     4501 aatttcttct ataaagtaac aaagggcccg tttaaactcg accagcttct gatggaatta
     4561 gaacttggca aaacaatact gagaatgaag tgtatgtgga acagaggctg ctgatctcgt
     4621 tcttcaggct atgaaactga cacatttgga aaccacagta cttagaacca caaagtggga
     4681 atcaagagaa aaacaatgat cccacgagag atctatagat ctatagatca tgagtgggag
     4741 gaatgagctg gcccttaatt tggttttgct tgtttaaatt atgatatcca actatgaaac
     4801 attatcataa agcaatagta aagagccttc agtaaagagc aggcatttat ctaatcccac
     4861 cccaccccca cccccgtagc tccaatcctt ccattcaaaa tgtaggtact ctgttctcac
     4921 ccttcttaac aaagtatgac aggaaaaact tccattttag tggacatctt tattgtttaa
     4981 tagatcatca atttctgcat cccgtcacca tcaactcatg ctcggttcat ctagtaacag
     5041 aaagtagctt gaatgtacaa gatcaatggc tggtgcttag ccctcccaca cataaccaga
     5101 gggcagcaat tcacgaatcc caactgccgt cggctgtcca tcactgtcct tcactatggc
     5161 tttgatccca ggatgcagat cgagaagcac ctgtcggcac cgtccgcagg ggctcaagat
     5221 gcccctgttc tcatttccga tcgcgacgat acaagtcagg ttgccagctg ccgcagcagc
     5281 agcagtgccc agcaccacga gttctgcaca aggtccccca gtaaaatgat atacattgac
     5341 accagtgaag atgcggccgt cgctagagag agctgcgctg gcgacgctgt agtcttcaga
     5401 gatggggatg ctgttgattg tagccgttgc tctttcaatg agggtggatt cttcttgaga
     5461 caaaggcatg gttgtggcca tattatcatc gtgtttttca aaggaaaacc acgtccccgt
     5521 ggttcggggg gcctagacgt ttttttaacc tcgactaaac acatgtaaag catgtgcacc
     5581 gaggccccag atcagatccc atacaatggg gtaccttctg ggcatccttc agccccttgt
     5641 tgaatacgct tgaggagagc catttgactc tttccacaac tatccaactc acaacgtggc
     5701 actggggttg tgccgccttt gcaggtgtat cttatacacg tggcttttgg ccgcagaggc
     5761 acctgtcgcc aggtgggggg ttccgctgcc tgcaaagggt cgctacagac gttgtttgtc
     5821 ttcaagaagc ttccagagga actgcttcct tcacgacatt caacagacct tgcattcctt
     5881 tggcgagagg ggaaagaccc ctaggaatgc tcgtcaagaa gacagggcca ggtttccggg
     5941 ccctcacatt gccaaaagac ggcaatatgg tggaaaataa catatagaca aacgcacacc
     6001 ggccttattc caagcggctt cggccagtaa cgttaggggg gggggaggga gagggggggg
     6061 gggcggaatt cgttaactta accggaaatc tccagagtag acagccagcg cgcgatctcg
     6121 ctgccgatca ggtccgggtt gtcttcttgc agcagattca gacccgggcc gatgtccaca
     6181 gccttgcagt taggcaggct tttggccagg cgagcggctt cggccggtgg gatcagaacg
     6241 cctggggtgc cccagaacag cagcttcggg acaggggact ggtgcagcca gtccatgtat
     6301 tcttcgacca gcgcgacgat gttcgctggc tcaccggcga ttggcagctc gtttgggaag
     6361 cgccacagtg gctcgcggtc aacaggattc aggaacggct cgcggtaatg gtccatctcg
     6421 acttcagtca gcgggcggac gacacccatc ggcagcgtac cctcgataaa aacgttctga
     6481 tcgatgatca gcttgcggcc gacgtcggtg gtgcggaagg cctggaaggt ctcgcgggca
     6541 aattctggcc attcgtccca ggtcgggata gggcggatga actccataaa tgcaatacct
     6601 ttgacgcgct ctggattgcg cttggcccag tggaaaccca gagcggagcc ccagtcgtga
     6661 atgaccagga cgacctcttc cagacccagg gcttcgatga aggcatccat gaagcggacg
     6721 tggtcgtcga agaaataacc caggtctggt ttgtcggatt tgcccatacc gatcaggtct
     6781 ggagcaatgc agcgatgggt cggtgcaaca tgcgggatga tgttgcgcca cacgtaggag
     6841 gaggtcgggt taccgtgcag gaacagcaca ggggtgccat cgcgcggacc aacatcgacg
     6901 tagtgcatgc gctcgcccag gacttccaca taatgggggt cgaatggaaa gccagtaccg
     6961 atttcggaac cgccaccaga gcctccacct ccacttccac ctccgcctga accacctcca
     7021 ccagatggat ccttagcgct ggtgtacttg gtgatggcct tagtaccctc ggacacggcg
     7081 tgcttggcca actccccagg cagcagcagg cgcacggccg tctggatctc cctggaggtg
     7141 atggtcgagc gcttgttgta atgcgccagg cgggaagcct cacctgcgat gcgctcgaaa
     7201 atgtcgttca caaacgaatt catgatgccc atggccttgg acgaaatgcc ggtgtcaggg
     7261 tggacctgct tcagaacctt gtacacatag atggaatagc tctccttgcg gctgcgcttg
     7321 cgcttcttgc cgcctttctt ctgcgcctta gtcaccgcct tcttggagcc ctttttcggg
     7381 gcgggagcag acttcgctgg ctctggcatc tcgagagatc aattctttgc caaaatgatg
     7441 agacagcaca ataaccagca cgttgcccag gagctgtagg aaaaagaaga aggcatgaac
     7501 atggttagca gaggctctag agccgccggt cacacgccag aagccgaacc ccgccctgcc
     7561 ccgtcccccc cgaaggcagc cgtccccccg cggacagccc cgaggctgga gagggagaag
     7621 gggacggcgg cgcggcgacg cacgaaggcc ctccccgccc atttccttcc tgccggcgcc
     7681 gcaccgcttc gccccgcgcc cgctagaggg ggtgcggcgg cgcctcccag atttcggctc
     7741 cgcacagatt tgggacaaag gaagtccctg cgccctctcg cacgattacc ataaaaggca
     7801 atggctgcgg ctcgccgcgc ctcgacagcc gccggcgctc cgggggccgc cgcgcccctc
     7861 ccccgagccc tccccggccc gaggcggccc cgccccgccc ggcaccccca cctgccgcca
     7921 ccccccgccc ggcacggcga gccccgcgcc acgccccgta cggagccccg cacccgaagc
     7981 cgggccgtgc tcagcaactc ggggaggggg gtgcaggggg ggttgcagcc cgaccgacgc
     8041 gcccacaccc cctgctcacc cccccacgca cacaccccgc acgcagcctt tgttcccctc
     8101 gcagcccccc ccgcaccgcg gggcaccgcc cccggccgcg ctcccctcgc gcacactgcg
     8161 gagcgcacaa agccccgcgc cgcgcccgca gcgctcacag ccgccgggca gcgcggagcc
     8221 gcacgcggcg ctccccacgc acacacacac gcacgcaccc cccgagccgc tccccccgca
     8281 caaagggccc tcccggagcc cctcaaggct t
//
