LOCUS       sequence-358073-        7244 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 7244)
  AUTHORS   Bruni R
  TITLE     Bruni Lab Empty Backbones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7244)
  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            "Unpublished"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: pNYCOMPS-N-Lic-His-FLAG
FEATURES             Location/Qualifiers
     source          1..7244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     113..135
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(129..320)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1129..2211)
                     /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     2173..2192
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2212..2289)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2495..2514
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2598..2617
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2598..2616
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2617..2641
                     /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             2656..2678
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2688..2711
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2712..2741
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2742..2762
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        2772..3204
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3205..3291,3292..4626)
                     /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             4706..4723
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4776..4794)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4790..4837
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4874..5329
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4961..4980)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5171..5192
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5421..6236)
                     /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(5421..6236)
                     /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     6144..6163
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6228..6247
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6358..6946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6847..6866
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7100..7117
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(7132..7244,1..27)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7197..7216)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
       61 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
      121 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
      181 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
      241 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
      301 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
      361 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
      421 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
      481 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
      541 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
      601 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
      661 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
      721 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
      781 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
      841 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
      901 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
      961 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     1021 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     1081 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     1141 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     1201 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     1261 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     1321 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     1381 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     1441 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     1501 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     1561 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     1621 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     1681 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     1741 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     1801 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     1861 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     1921 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     1981 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     2041 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     2101 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     2161 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     2221 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     2281 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     2341 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     2401 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     2461 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     2521 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     2581 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     2641 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatggat tataaagatg
     2701 atgatgataa acatcatcac catcaccatc accaccatca cgaaaacctg tattttcaat
     2761 cctacgtaat acgttcacta ttatttagtg aaatgagata ttatgatatt ttctgaattg
     2821 tgattaaaaa ggcaacttta tgcccatgca acagaaacta taaaaaatac agagaatgaa
     2881 aagaaacaga tagatttttt agttctttag gcccgtagtc tgcaaatcct tttatgattt
     2941 tctatcaaac aaaagaggaa aatagaccag ttgcaatcca aacgagagtc taatagaatg
     3001 aggtcgaaaa gtaaatcgcg cgggtttgtt actgataaag caggcaagac ctaaaatgtg
     3061 taaagggcaa agtgtatact ttggcgtcac cccttacata ttttaggtct ttttttattg
     3121 tgcgtaacta acttgccatc ttcaaacagg agggctggaa gaagcagacc gctaacacag
     3181 tacataaaaa aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta
     3241 ttaaccttta ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg
     3301 aaccaaaagc catataagga aacatacggc atttcccata ttacacgcca tgatatgctg
     3361 caaatccctg aacagcaaaa aaatgaaaaa tataaagttc ctgagttcga ttcgtccaca
     3421 attaaaaata tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac
     3481 actgacggca ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat
     3541 cctaaaaatg cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct
     3601 attgacagct ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat
     3661 gattctatcc taaaagacca aacacaagaa tggtcaggtt cagccacatt tacatctgac
     3721 ggaaaaatcc gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg
     3781 acaactgcac aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag
     3841 gattataaat caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc
     3901 gatgaaggca actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa
     3961 gataaaggcc acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa
     4021 ggcgaagaat ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa
     4081 gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct
     4141 ctcggtatga ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt
     4201 gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa
     4261 tggtacctgt tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac
     4321 gatatttaca tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac
     4381 aaaactggcc ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca
     4441 cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac
     4501 agaggattct acgcagacaa acaatcaacg tttgcgccta gcttcctgct gaacatcaaa
     4561 ggcaagaaaa catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac
     4621 aaataaaaac gcaaaagaaa atgccgatat cctattggca ttgacgtcag gtggcacata
     4681 ttacgtataa tattgagggc tcgagcacca ccaccaccac cactgagatc cggctgctaa
     4741 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     4801 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     4861 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4921 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4981 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5041 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5101 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5161 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5221 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5281 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5341 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5401 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     5461 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     5521 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     5581 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     5641 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     5701 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     5761 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     5821 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     5881 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     5941 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     6001 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     6061 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     6121 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     6181 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     6241 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     6301 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6361 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6421 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6481 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     6541 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6601 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6661 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6721 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6781 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6841 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6901 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6961 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     7021 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     7081 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     7141 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     7201 atctgctctg atgccgcata gttaagccag tatacactcc gcta
//
