LOCUS       sequence-366884-        5974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector of His-RanQ69L in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5974)
  AUTHORS   Mori M, Yao T, Mishina T, Endoh H, Tanaka M, Yonezawa N, Shimamoto 
            Y, Yonemura S, Yamagata K, Kitajima TS, Ikawa M
  TITLE     RanGTP and the actin cytoskeleton keep paternal and maternal 
            chromosomes apart during fertilization.
  JOURNAL   J Cell Biol. 2021 Oct 4;220(10). pii: 212591. doi: 
            10.1083/jcb.202012001. Epub 2021 Aug 23.
  PUBMED    34424312
REFERENCE   2  (bases 1 to 5974)
  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: "J Cell 
            Biol."; date: "2021-10-4"; volume: "220(10). pii: 212591. doi"; 
            pages: " 10.1083/jcb.202012001. Epub 2021 Aug 23"
COMMENT     Sequence Label: pET28b_RanWT
FEATURES             Location/Qualifiers
     source          1..5974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      48..503
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(135..154)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     345..366
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(595..1410)
                     /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(595..1410)
                     /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     1318..1337
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1402..1421
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1532..2120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2021..2040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2274..2291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2306..2445
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2371..2390)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2531..2553
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2547..2738)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3547..4629)
                     /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     4591..4610
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4630..4707)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4913..4932
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5016..5035
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5016..5034
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5035..5059
                     /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             5074..5096
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5115..5132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5142..5159
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5854..5871
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5924..5942)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(5938..5974,1..11)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
       61 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      121 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      181 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
      241 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
      301 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
      361 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
      421 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
      481 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
      541 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
      601 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
      661 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
      721 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
      781 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
      841 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
      901 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
      961 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1021 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     1081 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     1141 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     1201 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     1261 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     1321 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     1381 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     1441 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1501 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1561 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1621 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1681 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1741 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1801 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1861 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1921 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1981 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2041 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2101 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2161 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     2221 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     2281 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     2341 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2401 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     2461 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     2521 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     2581 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     2641 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     2701 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     2761 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     2821 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     2881 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     2941 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3001 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3061 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     3121 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     3181 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     3241 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     3301 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     3361 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     3421 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     3481 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     3541 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3601 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     3661 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     3721 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     3781 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     3841 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     3901 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     3961 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     4021 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     4081 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     4141 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     4201 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     4261 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     4321 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     4381 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     4441 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     4501 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     4561 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     4621 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     4681 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     4741 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     4801 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     4861 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     4921 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     4981 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     5041 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
     5101 ccatgggcag cagccatcat catcatcatc acagcagcgg cctggtgccg cgcggcagcc
     5161 atatggccgc ccagggagag ccgcaggtcc agttcaagct cgtcctggtg ggcgacggcg
     5221 gcaccgggaa gacaaccttc gtgaagcgcc acttgacggg cgagtttgag aagaagtatg
     5281 tagccaccct gggcgtggag gtgcacccgc tcgtcttcca taccaacaga ggacccatca
     5341 agttcaacgt gtgggacacg gccggccagg agaagttcgg gggcctgcgc gatggctact
     5401 acatccaagc ccagtgtgcc attataatgt ttgatgtaac ctcaagagtt acttacaaga
     5461 atgtacctaa ctggcataga gatctggtac gagtgtgtga aaacatcccc attgtattgt
     5521 gtggcaacaa agtggatatt aaagacagga aagtgaaggc aaaatctatt gtcttccacc
     5581 ggaagaagaa tcttcagtac tatgacattt ctgccaaaag taactacaac tttgaaaagc
     5641 ctttcctctg gcttgccaga aagctcattg gagatcctaa cttggagttt gttgccatgc
     5701 ctgctcttgc cccacctgag gtggtcatgg acccagcttt ggcagcacag tacgagcatg
     5761 atttagaggt tgctcagacg actgctctcc cagatgagga tgatgacctg tgagaattcg
     5821 agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     5881 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     5941 gcataacccc ttggggcctc taaacgggtc ttga
//
