LOCUS       sequence-361391-        7176 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 7176)
  AUTHORS   Bruni R
  TITLE     Bruni Lab Empty Backbones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7176)
  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            "Unpublished"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-C-Lic-His-FLAG
FEATURES             Location/Qualifiers
     source          1..7176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..79
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(73..264)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1073..2155)
                     /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     2117..2136
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2156..2233)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2439..2458
                     /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)."
     CDS             join(3064..3150,3151..4485)
                     /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             4522..4542
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4543..4572
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4582..4599
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4652..4670)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4666..4713
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4750..5205
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4837..4856)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5047..5068
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5297..6112)
                     /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(5297..6112)
                     /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     6020..6039
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6104..6123
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6234..6822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6723..6742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6976..6993
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7008..7147
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7073..7092)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
       61 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
      121 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
      181 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
      241 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
      301 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
      361 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
      421 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
      481 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
      541 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
      601 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
      661 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
      721 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
      781 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
      841 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
      901 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
      961 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     1021 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     1081 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     1141 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     1201 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     1261 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     1321 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     1381 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     1441 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     1501 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     1561 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     1621 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     1681 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     1741 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     1801 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     1861 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     1921 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     1981 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     2041 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     2101 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     2161 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     2221 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     2281 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     2341 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     2401 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     2461 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     2521 gagatcgatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     2581 aacaattccc ctctagacct taagaaggag atatactatg caggtcgttc actattatta
     2641 tatttagtga aatgagatat tatgatattt tctgaattgt gattaaaaag gcaactttat
     2701 gcccatgcaa cagaaactat aaaaaataca gagaatgaaa agaaacagat agatttttta
     2761 gttctttagg cccgtagtct gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa
     2821 atagaccagt tgcaatccaa acgagagtct aatagaatga ggtcgaaaag taaatcgcgc
     2881 gggtttgtta ctgataaagc aggcaagacc taaaatgtgt aaagggcaaa gtgtatactt
     2941 tggcgtcacc ccttacatat tttaggtctt tttttattgt gcgtaactaa cttgccatct
     3001 tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa ggagacatga
     3061 acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac taccgcactg
     3121 ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc atataaggaa
     3181 acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga acagcaaaaa
     3241 aatgaaaaat ataaagttcc tgagttcgat tcgtccacaa ttaaaaatat ctcttctgca
     3301 aaaggcctgg acgtttggga cagctggcca ttacaaaaca ctgacggcac tgtcgcaaac
     3361 tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc ggatgacaca
     3421 tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg gaaaaacgct
     3481 ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg attctatcct aaaagaccaa
     3541 acacaagaat ggtcaggttc agccacattt acatctgacg gaaaaatccg tttattctac
     3601 actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca agttaacgta
     3661 tcagcatcag acagctcttt gaacatcaac ggtgtagagg attataaatc aatctttgac
     3721 ggtgacggaa aaacgtatca aaatgtacag cagttcatcg atgaaggcaa ctacagctca
     3781 ggcgacaacc atacgctgag agatcctcac tacgtagaag ataaaggcca caaatactta
     3841 gtatttgaag caaacactgg aactgaagat ggctaccaag gcgaagaatc tttatttaac
     3901 aaagcatact atggcaaaag cacatcattc ttccgtcaag aaagtcaaaa acttctgcaa
     3961 agcgataaaa aacgcacggc tgagttagca aacggcgctc tcggtatgat tgagctaaac
     4021 gatgattaca cactgaaaaa agtgatgaaa ccgctgattg catctaacac agtaacagat
     4081 gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat ggtacctgtt cactgactcc
     4141 cgcggatcaa aaatgacgat tgacggcatt acgtctaacg atatttacat gcttggttat
     4201 gtttctaatt ctttaactgg cccatacaag ccgctgaaca aaactggcct tgtgttaaaa
     4261 atggatcttg atcctaacga tgtaaccttt acttactcac acttcgctgt acctcaagcg
     4321 aaaggaaaca atgtcgtgat tacaagctat atgacaaaca gaggattcta cgcagacaaa
     4381 caatcaacgt ttgcgcctag cttcctgctg aacatcaaag gcaagaaaac atctgttgtc
     4441 aaagacagca tccttgaaca aggacaatta acagttaaca aataaaaacg caaaagaaaa
     4501 ttttagtaag ccacctgcgc cgaaaacctc tattttcagg gacatcatca ccatcaccat
     4561 caccaccatc actaactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     4621 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     4681 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     4741 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4801 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4861 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4921 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4981 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5041 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     5101 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5161 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     5221 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5281 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     5341 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     5401 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     5461 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     5521 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     5581 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     5641 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     5701 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     5761 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     5821 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     5881 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     5941 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     6001 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     6061 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     6121 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     6181 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6241 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6301 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6361 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     6421 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6481 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6541 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6601 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6661 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6721 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6781 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6841 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6901 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     6961 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     7021 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     7081 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     7141 ggctgcgccc cgacacccgc caacacccgc tgacgc
//
