LOCUS       sequence-194159-        6249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CLIP-tagged Histone H2B in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6249)
  AUTHORS   Ana Egana
  TITLE     Egana Lab NEB plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6249)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCLIPf-H2B Control Plasmid
FEATURES             Location/Qualifiers
     source          1..6249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(87..106)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          89..318
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     CDS             complement(442..987)
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             complement(1042..1419)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(1461..1480)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1462..1480)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1525..1728)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1554..1574)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1729..2108
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2280..2299
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2374..2478
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2479..2547,2548..3339)
                     /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(2697..2716)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3510..4098
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3999..4018
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4252..4269
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4386..4407
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4422..4427,4428..4445,4446..4452)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4460..4476
                     /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     4465..4487
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4484..4500
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4484..4500
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4602..4723
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4667..4686
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4853..5656)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4964..4983)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5574..5593
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_feature    join(5679..5687,5688..5690,5691..6249,1..16)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5852..5871)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     6081..6098
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
ORIGIN
        1 gggggaggga gagggggggg ggggagaggg gcggaattgg ccgccctaga tgcatgctcg
       61 acctgcagtt ggacctggga gtggacacct gtggagagaa aggcaaagtg gatgtcattg
      121 tcactcaagt gtatggccag atctcaagcc tgccacacct caagcttgac aacaaaaaga
      181 ttgtcttttc tgaccagatg gacgcggcca ccctcaaagg catcaccgcg ggccaggtga
      241 atatcaaatc ctcctcgttt ttggaaactg acaatcttag cgcagaagtc atgcccgctt
      301 ttgagaggga gtactcaccc caacagctgg ccctcgcaga cagcgaatta attccagcac
      361 actggatcag ttatctatgc ggccggctta agcggccgct cattaattaa cctcgagttt
      421 aaacgcggat ccgcctgcag gacccagccc aggcttgccc agtctgtggc cctcgtgggc
      481 cagcagccac tctttcacgg cgagcccgcc caggtagggc cccacgtcgc tgtcgccctg
      541 caccacccgg tggcagggga tcagaatggg cacgggattt ccgtccaggg cggtgttcac
      601 ggcggcggtg gcggcgggat tgcccaccag ggcggccagg tggctctcgc tgatgacctc
      661 tccgaacttc accactttca gcagtttcca cagcacctgg cgggtaaagc tctcctgctg
      721 gaacactggg tggtgcaggg ctggcacagg gaactcctcg atggcctcag gctggtgaaa
      781 gtaggcgttg agccaggcgg tggcctggat cagtggctct ggtccgccca gcacggcggc
      841 tggggcaggc acttccacgg cgtcggcggc agatgttcct ttgcccagga agatgatacg
      901 gtgcaggccc tgttcgcacc cagacagttc cagcttgccc agagggctat ccagggtggt
      961 gcgcttcatt tcgcagtctt tgtccatggt gaattcaccg gtctgtacag atttaaatgc
     1021 tggcgcgccg atatcgctag ccttagcgct ggtgtacttg gtgatggcct tagtaccctc
     1081 ggacacggcg tgcttggcca actccccagg cagcagcagg cgcacggccg tctggatctc
     1141 cctggaggtg atggtcgagc gcttgttgta atgcgccagg cgggaagcct cacctgcgat
     1201 gcgctcgaaa atgtcgttca caaacgaatt catgatgccc atggccttgg acgaaatgcc
     1261 ggtgtcaggg tggacctgct tcagaacctt gtacacatag atggaatagc tctccttgcg
     1321 gctgcgcttg cgcttcttgc cgcctttctt ctgcgcctta gtcaccgcct tcttggagcc
     1381 ctttttcggg gcgggagcag acttcgctgg ctctggcatc aattcgatat cgatccgagc
     1441 tcggtaccaa gcttgggtct ccctatagtg agtcgtatta atttcgataa gccagtaagc
     1501 agtgggttct ctagttagcc agagagctct gcttatatag acctcccacc gtacacgcct
     1561 accgcccatt tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt
     1621 ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga
     1681 gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc
     1741 gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc
     1801 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg
     1861 atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca
     1921 atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg
     1981 ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc
     2041 catatatggg ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa
     2101 tcaatgtcaa cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct
     2161 aagcagattc ttcatgcaat tgtcggtcaa gccttgcctt gttgtagctt aaattttgct
     2221 cgcgcactac tcagcgacct ccaacacaca agcagggagc agatactggc ttaactatgc
     2281 ggcatcagag cagattgtac tgagagtgca ccatagggga tcgggagatc tcccgatccg
     2341 tcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2401 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2461 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2521 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2581 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2641 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2701 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2761 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2821 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2881 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2941 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3001 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3061 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3121 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3181 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3241 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3301 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3361 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3421 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3481 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3541 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3601 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3661 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3721 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3781 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3841 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3901 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3961 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4021 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4081 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4141 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4201 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4261 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     4321 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     4381 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     4441 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     4501 catgattacg ccaagctcta gctagaggtc gacggtatac agacatgata agatacattg
     4561 atgagtttgg acaaaccaca actagaatgc actcgacgcg ttaagataca ttgatgagtt
     4621 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     4681 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     4741 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     4801 ctacaaatgt ggtatggctg attatgatca gttatctaga gaattgatcc cctcagaaga
     4861 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
     4921 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
     4981 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
     5041 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
     5101 catcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
     5161 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
     5221 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
     5281 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
     5341 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
     5401 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
     5461 cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
     5521 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
     5581 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
     5641 caatggccga tcccatattg gctgcaggaa ttatcccggg ttgtggcaag cttatcatcg
     5701 tgtttttcaa aggaaaacca cgtccccgtg gttcgggggg cctagacgtt tttttaacct
     5761 cgactaaaca catgtaaagc atgtgcaccg aggccccaga tcagatccca tacaatgggg
     5821 taccttctgg gcatccttca gccccttgtt gaatacgctt gaggagagcc atttgactct
     5881 ttccacaact atccaactca caacgtggca ctggggttgt gccgcctttg caggtgtatc
     5941 ttatacacgt ggcttttggc cgcagaggca cctgtcgcca ggtggggggt tccgctgcct
     6001 gcaaagggtc gctacagacg ttgtttgtct tcaagaagct tccagaggaa ctgcttcctt
     6061 cacgacattc aacagacctt gcattccttt ggcgagaggg gaaagacccc taggaatgct
     6121 cgtcaagaag acagggccag gtttccgggc cctcacattg ccaaaagacg gcaatatggt
     6181 ggaaaataac atatagacaa acgcacaccg gccttattcc aagcggcttc ggccagtaac
     6241 gttaggggg
//
