LOCUS       Exported                5673 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression of His-tagged Ran in the pET28 protein 
            expression backbone.
ACCESSION   .
VERSION     .
KEYWORDS    pET28-NTF2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5673)
  TITLE     Simon lab plasmids
REFERENCE   2  (bases 1 to 5673)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5673
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     61..83
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(77..268)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1077..2159)
                     /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     2121..2140
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2160..2237)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2443..2462
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2546..2565
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2546..2564
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2565..2589
                     /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             2604..2626
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2645..2662
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2672..2689
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3083..3100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3167..3214
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3251..3706
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3338..3357)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3548..3569
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3798..4613)
                     /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     4521..4540
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4605..4624
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4735..5323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5224..5243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5477..5494
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5509..5648
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5574..5593)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
       61 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
      121 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
      181 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
      241 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
      301 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
      361 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
      421 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
      481 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
      541 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
      601 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
      661 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
      721 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
      781 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
      841 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
      901 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
      961 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     1021 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     1081 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1141 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     1201 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     1261 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     1321 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     1381 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     1441 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     1501 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     1561 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     1621 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     1681 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     1741 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     1801 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     1861 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     1921 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     1981 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     2041 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     2101 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     2161 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     2221 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     2281 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     2341 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     2401 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     2461 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     2521 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     2581 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata ccatgggcag
     2641 cagccatcat catcatcatc acagcagcgg cctggtgccg cgcggcagcc atatgggcga
     2701 caaaccgatc tgggagcaaa tcggcagcag ctttatccaa cactactacc aactgttcga
     2761 caatgaccgc acccaactgg gtgcgatcta cattgacgcg agctgcctga cctgggaggg
     2821 tcagcaattc cagggcaagg cggcgatcgt ggaaaaactg agcagcctgc cgtttcagaa
     2881 gatccaacac agcattaccg cgcaggacca ccaaccgacc ccggatagct gcatcattag
     2941 catggtggtt ggtcagctga aggcggacga ggacccgatc atgggcttcc accaaatgtt
     3001 tctgctgaaa aacattaacg atgcgtgggt ttgcaccaat gatatgttcc gtctggcgct
     3061 gcacaacttc ggctaactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     3121 agcccgaaag gaagctgagt tggctgctgc caccgccgag caataactag cataacccct
     3181 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     3241 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3301 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3361 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3421 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3481 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3541 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3601 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3661 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     3721 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3781 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactt caatttattc
     3841 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     3901 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     3961 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     4021 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     4081 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     4141 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     4201 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     4261 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     4321 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     4381 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     4441 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     4501 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     4561 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     4621 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     4681 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4741 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4801 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4861 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     4921 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4981 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5041 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5101 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5161 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5221 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5281 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5341 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5401 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     5461 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     5521 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     5581 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     5641 tggctgcgcc ccgacacccg ccaacacccg ctg
//
