LOCUS       sequence-359617-        6655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial protein expression vector for GFP-tagged HP1alpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6655)
  AUTHORS   Erdel F, Rademacher A, Vlijm R, Tunnermann J, Frank L, Weinmann R, 
            Schweigert E, Yserentant K, Hummert J, Bauer C, Schumacher S, Al 
            Alwash A, Normand C, Herten DP, Engelhardt J, Rippe K
  TITLE     Mouse Heterochromatin Adopts Digital Compaction States without 
            Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation.
  JOURNAL   Mol Cell. 2020 Apr 16;78(2):236-249.e7. doi: 
            10.1016/j.molcel.2020.02.005. Epub 2020 Feb 25.
  PUBMED    32101700
REFERENCE   2  (bases 1 to 6655)
  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"; doi: 
            "10.1016/j.molcel.2020.02.005"; journalName: "Mol Cell"; date: 
            "2020-04-16- 16"; volume: "78"; issue: "2"; pages: "236-249.e7"
COMMENT     Sequence Label: pET28-GFP-mHP1alpha
FEATURES             Location/Qualifiers
     source          1..6655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      6..461
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(93..112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     303..324
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(553..1368)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(553..1368)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1276..1295
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1360..1379
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1490..2078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1979..1998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2232..2249
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2264..2403
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2329..2348)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2489..2511
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2505..2696)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3505..4587)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4549..4568
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4588..4665)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4871..4890
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4974..4993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4974..4992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4993..5017
                     /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)."
     RBS             5032..5054
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5073..5090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5100..5117
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(5148..5150,5151..5153,5154..5864)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5148..5150,5151..5153,5154..5864)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5193..5214)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5454..5473)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5801..5822
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6493..6510
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6563..6581)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6577..6624
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
       61 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
      121 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
      181 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
      241 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
      301 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
      361 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
      421 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
      481 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      541 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
      601 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
      661 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
      721 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
      781 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
      841 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
      901 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
      961 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     1021 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     1081 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     1141 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     1201 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     1261 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     1321 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     1381 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     1441 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1501 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1561 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1621 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1681 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1741 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1801 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1861 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1921 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     1981 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2041 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2101 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2161 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2221 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     2281 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     2341 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     2401 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     2461 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     2521 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     2581 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     2641 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     2701 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     2761 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     2821 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     2881 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     2941 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     3001 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     3061 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     3121 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     3181 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     3241 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     3301 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     3361 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     3421 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     3481 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3541 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     3601 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     3661 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     3721 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     3781 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     3841 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     3901 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     3961 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4021 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4081 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4141 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4201 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     4261 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     4321 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     4381 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     4441 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     4501 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     4561 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     4621 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     4681 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     4741 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     4801 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     4861 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     4921 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     4981 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     5041 ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc
     5101 tggtgccgcg cggcagccat atggctagcg ctaccggtcg ccaccttatg gtgagcaagg
     5161 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     5221 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     5281 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     5341 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     5401 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     5461 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     5521 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     5581 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     5641 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     5701 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     5761 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     5821 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga atgggaaaga
     5881 agaccaagag gacagccgac agctcttctt cagaggatga ggaggaatat gtggtggaaa
     5941 aggtgttgga caggcgcatg gttaaggggc aagtggaata tctgttgaag tggaaaggct
     6001 tttctgagga gcacaatact tgggaacctg agaagaactt ggattgtcct gaactaattt
     6061 ctgagtttat gaaaaagtat aagaagatga aggagggtga aaacaataag cccagggaga
     6121 aatcagaagg aaacaagagg aaatccagtt tctccaacag cgctgatgat attaaatcta
     6181 aaaaaaagag agagcaaagc aatgatatcg ctcggggctt tgagagagga ctggaaccag
     6241 aaaagatcat cggagcaaca gattcctgcg gtgacttaat gttcttaatg aaatggaaag
     6301 acacagatga agctgacctg gttcttgcaa aagaagctaa cgtgaagtgt ccacagattg
     6361 tgatagcatt ttatgaagag agactgacgt ggcacgcata tccagaggat gcggaaaaca
     6421 aagaaaaaga aagcgcgaag agctaaggat ccgaattcga gctccgtcga caagcttgcg
     6481 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     6541 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     6601 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcga
//
