LOCUS       sequence-366883-        5974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector of His-RanT24N 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_RanT24N
FEATURES             Location/Qualifiers
     source          1..5974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     222..241
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(261..338)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     544..563
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     647..666
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        647..665
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    666..690
                     /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             705..727
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             746..763
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             773..790
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1485..1502
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1555..1573)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1569..1616
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1653..2108
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1740..1759)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1950..1971
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2200..3015)
                     /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(2200..3015)
                     /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     2923..2942
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3007..3026
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3137..3725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3626..3645
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3879..3896
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3911..4050
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3976..3995)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4136..4158
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4152..4343)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5152..5974,1..260))
                     /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"
ORIGIN
        1 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
       61 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
      121 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
      181 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
      241 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
      301 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
      361 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
      421 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
      481 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
      541 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
      601 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
      661 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
      721 aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc
      781 gcgcggcagc catatggccg cccagggaga gccgcaggtc cagttcaagc tcgtcctggt
      841 gggcgacggc ggcaccggga agaatacctt cgtgaagcgc cacttgacgg gcgagtttga
      901 gaagaagtat gtagccaccc tgggcgtgga ggtgcacccg ctcgtcttcc ataccaacag
      961 aggacccatc aagttcaacg tgtgggacac ggccggccag gagaagttcg ggggcctgcg
     1021 cgatggctac tacatccaag cccagtgtgc cattataatg tttgatgtaa cctcaagagt
     1081 tacttacaag aatgtaccta actggcatag agatctggta cgagtgtgtg aaaacatccc
     1141 cattgtattg tgtggcaaca aagtggatat taaagacagg aaagtgaagg caaaatctat
     1201 tgtcttccac cggaagaaga atcttcagta ctatgacatt tctgccaaaa gtaactacaa
     1261 ctttgaaaag cctttcctct ggcttgccag aaagctcatt ggagatccta acttggagtt
     1321 tgttgccatg cctgctcttg ccccacctga ggtggtcatg gacccagctt tggcagcaca
     1381 gtacgagcat gatttagagg ttgctcagac gactgctctc ccagatgagg atgatgacct
     1441 gtgagaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
     1501 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1561 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1621 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1681 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1741 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1801 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1861 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1921 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1981 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2041 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2101 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2161 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     2221 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     2281 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     2341 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     2401 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     2461 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     2521 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     2581 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     2641 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     2701 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     2761 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     2821 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     2881 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     2941 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     3001 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     3061 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3121 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3181 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3241 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3301 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3361 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3421 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3481 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3541 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3601 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3661 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3721 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3781 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3841 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3901 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3961 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4021 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     4081 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4141 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4201 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4261 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4321 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4381 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4441 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4501 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4561 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4621 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4681 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4741 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4801 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4861 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4921 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4981 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     5041 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     5101 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     5161 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5221 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5281 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     5341 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5401 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     5461 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5521 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5581 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5641 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5701 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5761 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5821 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5881 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5941 catcgacacc accacgctgg cacccagttg atcg
//
