LOCUS       sequence-316745-        5677 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 5677)
  TITLE     Simon lab plasmids
REFERENCE   2  (bases 1 to 5677)
  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-NTF2
FEATURES             Location/Qualifiers
     source          1..5677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     45..67
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(61..252)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1061..2143)
                     /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     2105..2124
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2144..2221)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2427..2446
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2530..2549
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2530..2548
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2549..2573
                     /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             2588..2610
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2629..2646
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2656..2673
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3067..3084
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3137..3155)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3151..3198
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3235..3690
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3322..3341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3532..3553
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3783..4598)
                     /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(3783..4598)
                     /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     4506..4525
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4590..4609
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4720..5308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5209..5228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5462..5479
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5494..5636
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5562..5581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
       61 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
      121 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
      181 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
      241 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
      301 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
      361 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
      421 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
      481 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
      541 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
      601 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
      661 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
      721 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
      781 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
      841 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
      901 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
      961 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     1021 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1081 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1141 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     1201 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     1261 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     1321 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     1381 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     1441 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     1501 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     1561 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     1621 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     1681 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     1741 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     1801 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     1861 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     1921 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     1981 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     2041 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     2101 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     2161 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     2221 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     2281 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     2341 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     2401 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     2461 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     2521 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     2581 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     2641 catcacagca gcggcctggt gccgcgcggc agccatatgg gcgacaaacc gatctgggag
     2701 caaatcggca gcagctttat ccaacactac taccaactgt tcgacaatga ccgcacccaa
     2761 ctgggtgcga tctacattga cgcgagctgc ctgacctggg agggtcagca attccagggc
     2821 aaggcggcga tcgtggaaaa actgagcagc ctgccgtttc agaagatcca acacagcatt
     2881 accgcgcagg accaccaacc gaccccggat agctgcatca ttagcatggt ggttggtcag
     2941 ctgaaggcgg acgaggaccc gatcatgggc ttccaccaaa tgtttctgct gaaaaacatt
     3001 aacgatgcgt gggtttgcac caatgatatg ttccgtctgg cgctgcacaa cttcggctaa
     3061 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     3121 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     3181 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     3241 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3301 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3361 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3421 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3481 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3541 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3601 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3661 attttaacaa aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg
     3721 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     3781 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     3841 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     3901 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     3961 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     4021 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     4081 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     4141 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     4201 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     4261 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     4321 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     4381 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     4441 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     4501 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     4561 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     4621 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     4681 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4741 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4801 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4861 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4921 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4981 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5041 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5101 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5161 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5221 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5281 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5341 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5401 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5461 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     5521 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     5581 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     5641 gacacccgcc aacacccgct gacgcgccct gacgggc
//
