LOCUS       sequence-321640-        5940 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His-tagged Ran in the pET28 protein 
            expression backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5940)
  TITLE     Simon lab plasmids
REFERENCE   2  (bases 1 to 5940)
  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"
COMMENT     Sequence Label: pET28-RAN
FEATURES             Location/Qualifiers
     source          1..5940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     102..124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(118..309)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1118..2200)
                     /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     2162..2181
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2201..2278)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2484..2503
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2587..2606
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2587..2605
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2606..2630
                     /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             2645..2667
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2686..2703
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2713..2730
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3391..3408
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3461..3479)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3475..3522
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3559..4014
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3646..3665)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3856..3877
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4106..4921)
                     /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
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLKFHLMLDEFF"
     primer_bind     4829..4848
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4913..4932
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5043..5631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5532..5551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5785..5802
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(5817..5940,1..16)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5882..5901)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
       61 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
      121 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
      181 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
      241 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
      301 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
      361 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
      421 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
      481 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
      541 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
      601 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
      661 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
      721 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
      781 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
      841 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
      901 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
      961 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     1021 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     1081 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1141 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1201 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     1261 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     1321 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     1381 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     1441 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     1501 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     1561 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     1621 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     1681 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     1741 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     1801 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     1861 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     1921 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     1981 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     2041 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     2101 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     2161 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     2221 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     2281 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     2341 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     2401 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     2461 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     2521 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     2581 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     2641 taattttgtt taactttaag aaggagatat accatgggca gcagccatca tcatcatcat
     2701 cacagcagcg gcctggtgcc gcgcggcagc catatggctg cgcagggaga gccccaggtc
     2761 cagttcaaac ttgtattggt tggtgatggt ggtactggaa aaacgacctt cgtgaaacgt
     2821 catttgactg gtgaatttga gaagaagtat gtagccacct tgggtgttga ggttcatccc
     2881 ctagtgttcc acaccaacag aggacctatt aagttcaatg tatgggacac agccggccag
     2941 gagaaattcg gtggactgag agatggctat tatatccaag cccagtgtgc catcataatg
     3001 tttgatgtaa catcgagagt tacttacaag aatgtgccta actggcatag agatctggta
     3061 cgagtgtgtg aaaacatccc cattgtgttg tgtggcaaca aagtggatat taaggacagg
     3121 aaagtgaagg cgaaatccat tgtcttccac cgaaagaaga atcttcagta ctacgacatt
     3181 tctgccaaaa gtaactacaa ctttgaaaag cccttcctct ggcttgctag gaagctcatt
     3241 ggagacccta acttggaatt tgttgccatg cctgctctcg ccccaccaga agttgtcatg
     3301 gacccagctt tggcagcaca gtatgagcac gacttagagg ttgctcagac aactgctctc
     3361 ccggatgagg atgatgacct gtgactcgag caccaccacc accaccactg agatccggct
     3421 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     3481 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     3541 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3601 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3661 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3721 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3781 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3841 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3901 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3961 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     4021 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4081 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaacttca
     4141 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     4201 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     4261 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     4321 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     4381 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     4441 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     4501 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     4561 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     4621 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     4681 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     4741 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     4801 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     4861 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     4921 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     4981 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5041 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5101 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5161 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     5221 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5281 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5341 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5401 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5461 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5521 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5581 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5641 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5701 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5761 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     5821 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     5881 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
//
