LOCUS       sequence-366885-        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_Q69L
FEATURES             Location/Qualifiers
     source          1..5974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     266..287
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(516..1331)
                     /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(516..1331)
                     /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     1239..1258
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1323..1342
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1453..2041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1942..1961
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2195..2212
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2227..2366
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2292..2311)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2452..2474
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2468..2659)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3468..4550)
                     /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     4512..4531
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4551..4628)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4834..4853
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4937..4956
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4937..4955
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4956..4980
                     /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             4995..5017
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5036..5053
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5063..5080
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5775..5792
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5845..5863)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5859..5906
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5943..5974,1..424)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
       61 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      121 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      181 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
      241 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
      301 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
      361 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
      421 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      481 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
      541 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
      601 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
      661 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
      721 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
      781 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
      841 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
      901 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
      961 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1021 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     1081 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1141 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1201 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     1261 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     1321 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     1381 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1441 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1501 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1561 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     1621 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1681 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1741 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1801 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1861 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1921 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     1981 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2041 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     2101 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     2161 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     2221 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     2281 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     2341 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     2401 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2461 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     2521 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     2581 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     2641 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     2701 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     2761 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     2821 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     2881 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     2941 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     3001 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     3061 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     3121 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     3181 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     3241 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     3301 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     3361 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     3421 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     3481 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3541 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     3601 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     3661 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     3721 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     3781 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     3841 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     3901 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     3961 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     4021 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     4081 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     4141 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     4201 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     4261 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     4321 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     4381 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     4441 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     4501 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     4561 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     4621 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     4681 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     4741 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     4801 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     4861 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     4921 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     4981 cctctagaaa taattttgtt taactttaag aaggagatat accatgggca gcagccatca
     5041 tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatggccg cccagggaga
     5101 gccgcaggtc cagttcaagc tcgtcctggt gggcgacggc ggcaccggga agacaacctt
     5161 cgtgaagcgc cacttgacgg gcgagtttga gaagaagtat gtagccaccc tgggcgtgga
     5221 ggtgcacccg ctcgtcttcc ataccaacag aggacccatc aagttcaacg tgtgggacac
     5281 ggccggcctg gagaagttcg ggggcctgcg cgatggctac tacatccaag cccagtgtgc
     5341 cattataatg tttgatgtaa cctcaagagt tacttacaag aatgtaccta actggcatag
     5401 agatctggta cgagtgtgtg aaaacatccc cattgtattg tgtggcaaca aagtggatat
     5461 taaagacagg aaagtgaagg caaaatctat tgtcttccac cggaagaaga atcttcagta
     5521 ctatgacatt tctgccaaaa gtaactacaa ctttgaaaag cctttcctct ggcttgccag
     5581 aaagctcatt ggagatccta acttggagtt tgttgccatg cctgctcttg ccccacctga
     5641 ggtggtcatg gacccagctt tggcagcaca gtacgagcat gatttagagg ttgctcagac
     5701 gactgctctc ccagatgagg atgatgacct gtgagaattc gagctccgtc gacaagcttg
     5761 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     5821 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     5881 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     5941 ggacgcgccc tgtagcggcg cattaagcgc ggcg
//
