LOCUS       sequence-326616-        5671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET based vector for protein expression in E.coli for targets fused 
            with N-terminal Flag and His.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5671)
  AUTHORS   Bruni R, Kloss B
  TITLE     High-throughput cloning and expression of integral membrane proteins
            in Escherichia coli.
  JOURNAL   Curr Protoc Protein Sci. 2013 Nov 5;74:29.6.1-29.6.34. doi: 
            10.1002/0471140864.ps2906s74.
  PUBMED    24510647
REFERENCE   2  (bases 1 to 5671)
  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: "Curr Protoc
            Protein Sci."; date: "2013-11-5"; volume: "74:29.6.1-29.6.34. doi"; 
            pages: " 10.1002/0471140864.ps2906s74"
COMMENT     Sequence Label: pN23-LIC-His-FLAG
FEATURES             Location/Qualifiers
     source          1..5671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     456..475
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     709..726
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    741..880
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(806..825)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     966..988
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(982..1173)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1682..1701
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1682..1700
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1732..1754
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1762..1794
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     RBS             1803..1825
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1835..1858
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1859..1888
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1889..1909
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        1919..2351
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(2352..2438,2439..3773)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             3853..3870
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3923..3941)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3937..3984
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4021..4476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4108..4127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4318..4339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4502..4606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4607..4675,4676..5467)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4825..4844)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5638..5671,1..555)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
       61 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
      121 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
      181 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      241 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      301 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
      361 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      421 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      481 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      541 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      601 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      661 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      721 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
      781 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
      841 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
      901 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
      961 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     1021 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     1081 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     1141 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     1201 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     1261 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     1321 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     1381 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     1441 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     1501 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     1561 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     1621 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatctcga tcccgcgaaa
     1681 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagaaataa ttttgtttaa
     1741 ctttaagaag gagatataca tatggctagc atgactggtg gacagcaaat gggtcgggat
     1801 catttgttta actttaagaa ggagatatac catggattat aaagatgatg atgataaaca
     1861 tcatcaccat caccatcacc accatcacga aaacctgtat tttcaatcct acgtaatacg
     1921 ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc
     1981 aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag
     2041 attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa
     2101 agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta
     2161 aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt
     2221 gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact
     2281 tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg
     2341 agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta
     2401 ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat
     2461 ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac
     2521 agcaaaaaaa tgaaaaatat aaagttcctg agttcgattc gtccacaatt aaaaatatct
     2581 cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacact gacggcactg
     2641 tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg
     2701 atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga
     2761 aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa
     2821 aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt
     2881 tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag
     2941 ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa
     3001 tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact
     3061 acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca
     3121 aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt
     3181 tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac
     3241 ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg
     3301 agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag
     3361 taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca
     3421 ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc
     3481 ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg
     3541 tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac
     3601 ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg
     3661 cagacaaaca atcaacgttt gcgcctagct tcctgctgaa catcaaaggc aagaaaacat
     3721 ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca
     3781 aaagaaaatg ccgatatcct attggcattg acgtcaggtg gcacatatta cgtataatat
     3841 tgagggctcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     3901 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     3961 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     4021 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4081 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4141 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4201 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     4261 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4321 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4381 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4441 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     4501 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4561 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4621 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4681 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4741 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4801 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4861 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4921 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4981 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5041 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5101 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     5161 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5221 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5281 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5341 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5401 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5461 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5521 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5581 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5641 agatcctttt tttctgcgcg taatctgctg c
//
