LOCUS       sequence-326615-        5589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET based vector for protein expression in E.coli for targets fused 
            with C-terminal Flag and His.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5589)
  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 5589)
  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: pC23-LIC-His-FLAG
FEATURES             Location/Qualifiers
     source          1..5589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..63
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(57..248)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     757..776
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        757..775
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1267..1353,1354..2688)
                     /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             2762..2782
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2783..2806
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2807..2836
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2846..2863
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2916..2934)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2930..2977
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3014..3469
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3101..3120)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3311..3332
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3495..3599
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3600..3668,3669..4460)
                     /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(3818..3837)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4631..5219
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5120..5139
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5373..5390
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5405..5544
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5470..5489)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
       61 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
      121 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
      181 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
      241 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
      301 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
      361 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
      421 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
      481 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
      541 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
      601 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
      661 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag
      721 gacccaacgc tgcccgagat ctcgatcccg cgaaattaat acgactcact atagggagtc
      781 cacaacggtt tccctctaga ccttaagaag gagatatact atgcaggtcg ttcactatta
      841 ttatatttag tgaaatgaga tattatgata ttttctgaat tgtgattaaa aaggcaactt
      901 tatgcccatg caacagaaac tataaaaaat acagagaatg aaaagaaaca gatagatttt
      961 ttagttcttt aggcccgtag tctgcaaatc cttttatgat tttctatcaa acaaaagagg
     1021 aaaatagacc agttgcaatc caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg
     1081 cgcgggtttg ttactgataa agcaggcaag acctaaaatg tgtaaagggc aaagtgtata
     1141 ctttggcgtc accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca
     1201 tcttcaaaca ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca
     1261 tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca
     1321 ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag
     1381 gaaacatacg gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa
     1441 aaaaatgaaa aatataaagt tcctgagttc gattcgtcca caattaaaaa tatctcttct
     1501 gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa acactgacgg cactgtcgca
     1561 aactatcacg gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac
     1621 acatcgattt acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac
     1681 gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac
     1741 caaacacaag aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc
     1801 tacactgatt tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac
     1861 gtatcagcat cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt
     1921 gacggtgacg gaaaaacgta tcaaaatgta cagcagttca tcgatgaagg caactacagc
     1981 tcaggcgaca accatacgct gagagatcct cactacgtag aagataaagg ccacaaatac
     2041 ttagtatttg aagcaaacac tggaactgaa gatggctacc aaggcgaaga atctttattt
     2101 aacaaagcat actatggcaa aagcacatca ttcttccgtc aagaaagtca aaaacttctg
     2161 caaagcgata aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat gattgagcta
     2221 aacgatgatt acacactgaa aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca
     2281 gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca aatggtacct gttcactgac
     2341 tcccgcggat caaaaatgac gattgacggc attacgtcta acgatattta catgcttggt
     2401 tatgtttcta attctttaac tggcccatac aagccgctga acaaaactgg ccttgtgtta
     2461 aaaatggatc ttgatcctaa cgatgtaacc tttacttact cacacttcgc tgtacctcaa
     2521 gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa acagaggatt ctacgcagac
     2581 aaacaatcaa cgtttgcgcc tagcttcctg ctgaacatca aaggcaagaa aacatctgtt
     2641 gtcaaagaca gcatccttga acaaggacaa ttaacagtta acaaataaaa acgcaaaaga
     2701 aaatgccgat atcctattgg cattgacgtc aggtgctggc ttttagtaag ccacctgcgc
     2761 cgaaaacctc tattttcagg gagattataa agatgatgat gataaacatc atcaccatca
     2821 ccatcaccac catcactaac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     2881 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     2941 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     3001 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3061 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3121 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3181 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3241 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3301 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3361 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3421 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3481 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3541 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3601 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3661 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3721 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3781 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3841 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3901 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3961 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4021 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4081 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4141 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4201 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4261 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggatctc
     4321 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4381 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4441 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4501 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4561 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4621 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4681 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4741 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4801 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4861 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4921 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4981 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5041 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5101 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5161 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5221 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5281 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5341 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5401 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     5461 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     5521 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     5581 cgggcttgt
//
