LOCUS       sequence-361393-        5665 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 STREP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5665)
  AUTHORS   Bruni R
  TITLE     Bruni Lab Empty Backbones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5665)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pN23-LIC-FLAG-STREP
FEATURES             Location/Qualifiers
     source          1..5665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     500..519
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        500..518
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             550..572
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             580..612
                     /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             621..643
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             653..676
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             677..700
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             701..721
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        731..1163
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1164..1250,1251..2585)
                     /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             2665..2682
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2735..2753)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2749..2796
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2833..3288
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2920..2939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3130..3151
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3314..3418
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3419..3487,3488..4279)
                     /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(3637..3656)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4450..5038
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4939..4958
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5192..5209
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5224..5363
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5289..5308)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5449..5471
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5465..5656)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
       61 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
      121 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
      181 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
      241 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
      301 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
      361 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
      421 ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga
      481 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
      541 agaaataatt ttgtttaact ttaagaagga gatatacata tggctagcat gactggtgga
      601 cagcaaatgg gtcgggatca tttgtttaac tttaagaagg agatatacca tggattataa
      661 agatgatgat gataaatgga gccacccgca gttcgaaaaa gaaaacctgt attttcaatc
      721 ctacgtaata cgttcactat tatttagtga aatgagatat tatgatattt tctgaattgt
      781 gattaaaaag gcaactttat gcccatgcaa cagaaactat aaaaaataca gagaatgaaa
      841 agaaacagat agatttttta gttctttagg cccgtagtct gcaaatcctt ttatgatttt
      901 ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa acgagagtct aatagaatga
      961 ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc aggcaagacc taaaatgtgt
     1021 aaagggcaaa gtgtatactt tggcgtcacc ccttacatat tttaggtctt tttttattgt
     1081 gcgtaactaa cttgccatct tcaaacagga gggctggaag aagcagaccg ctaacacagt
     1141 acataaaaaa ggagacatga acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat
     1201 taacctttac taccgcactg ctggcaggag gcgcaactca agcgtttgcg aaagaaacga
     1261 accaaaagcc atataaggaa acatacggca tttcccatat tacacgccat gatatgctgc
     1321 aaatccctga acagcaaaaa aatgaaaaat ataaagttcc tgagttcgat tcgtccacaa
     1381 ttaaaaatat ctcttctgca aaaggcctgg acgtttggga cagctggcca ttacaaaaca
     1441 ctgacggcac tgtcgcaaac tatcacggct accacatcgt ctttgcatta gccggagatc
     1501 ctaaaaatgc ggatgacaca tcgatttaca tgttctatca aaaagtcggc gaaacttcta
     1561 ttgacagctg gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg
     1621 attctatcct aaaagaccaa acacaagaat ggtcaggttc agccacattt acatctgacg
     1681 gaaaaatccg tttattctac actgatttct ccggtaaaca ttacggcaaa caaacactga
     1741 caactgcaca agttaacgta tcagcatcag acagctcttt gaacatcaac ggtgtagagg
     1801 attataaatc aatctttgac ggtgacggaa aaacgtatca aaatgtacag cagttcatcg
     1861 atgaaggcaa ctacagctca ggcgacaacc atacgctgag agatcctcac tacgtagaag
     1921 ataaaggcca caaatactta gtatttgaag caaacactgg aactgaagat ggctaccaag
     1981 gcgaagaatc tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag
     2041 aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc
     2101 tcggtatgat tgagctaaac gatgattaca cactgaaaaa agtgatgaaa ccgctgattg
     2161 catctaacac agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat
     2221 ggtacctgtt cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg
     2281 atatttacat gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca
     2341 aaactggcct tgtgttaaaa atggatcttg atcctaacga tgtaaccttt acttactcac
     2401 acttcgctgt acctcaagcg aaaggaaaca atgtcgtgat tacaagctat atgacaaaca
     2461 gaggattcta cgcagacaaa caatcaacgt ttgcgcctag cttcctgctg aacatcaaag
     2521 gcaagaaaac atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaaca
     2581 aataaaaacg caaaagaaaa tgccgatatc ctattggcat tgacgtcagg tggcacatat
     2641 tacgtataat attgagggct cgagcaccac caccaccacc actgagatcc ggctgctaac
     2701 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     2761 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     2821 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2881 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2941 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3001 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3061 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3121 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3181 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3241 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     3301 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3361 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3421 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3481 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3541 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3601 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3661 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3721 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3781 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3841 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3901 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3961 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4021 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4081 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4141 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4201 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4261 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4321 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4381 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4441 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4501 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4561 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4621 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4681 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4741 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4801 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4861 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4921 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4981 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5041 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5101 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5161 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5221 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     5281 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     5341 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     5401 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5461 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     5521 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     5581 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     5641 tttcctgttt ggtcactgat gcctc
//
