LOCUS       sequence-67089-e        6976 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX cloning E.coli expression vector with T7 promoter and N-terminal 
            10x His tag and 3C protease cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6976)
  AUTHORS   Geertsma ER, Dutzler R
  TITLE     A versatile and efficient high-throughput cloning tool for 
            structural biology.
  JOURNAL   Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25.
  PUBMED    21410291
REFERENCE   2  (bases 1 to 6976)
  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: 
            "Biochemistry."; date: "2011-04-19"; volume: "50(15)"; pages: 
            "3272-8. Epub 2011 Mar 25"
COMMENT     Sequence Label: p7XNH3
FEATURES             Location/Qualifiers
     source          1..6976
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             6..23
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(76..94)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      90..137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      174..629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(261..280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     471..492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(721..1536)
                     /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(721..1536)
                     /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     1444..1463
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1528..1547
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1658..2246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2147..2166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2400..2417
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2432..2574
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2500..2519)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2660..2682
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2676..2867)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3676..4758)
                     /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     4720..4739
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4759..4836)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5042..5061
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5145..5164
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5145..5163
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5164..5188
                     /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             5203..5225
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5236..5265
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5266..5289
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(5338..5643)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(5440..5459)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5985..6644)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6559..6578
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6698..6704,6705..6722,6723..6728))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(6753..6877)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(6896..6916)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6920..6940)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
       61 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      121 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      181 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      241 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      301 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      361 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      421 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      481 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      541 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      601 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
      661 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
      721 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
      781 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
      841 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
      901 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
      961 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     1021 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1081 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1141 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1201 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     1261 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     1321 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     1381 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     1441 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     1501 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     1561 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     1621 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1681 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1741 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1801 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1861 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1921 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1981 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2041 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2101 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2161 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2221 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2281 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2341 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2401 gcgagtcagt gagcgaggaa gcggatcagc gcctgatgcg gtattttctc cttacgcatc
     2461 tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc
     2521 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     2581 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2641 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2701 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     2761 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     2821 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     2881 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     2941 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3001 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3061 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3121 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3181 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3241 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     3301 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     3361 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     3421 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     3481 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     3541 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     3601 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     3661 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3721 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     3781 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     3841 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     3901 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     3961 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4021 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4081 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4141 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4201 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4261 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4321 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     4381 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     4441 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     4501 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4561 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     4621 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     4681 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     4741 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4801 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     4861 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     4921 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     4981 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     5041 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     5101 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     5161 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     5221 ggagatatac atatgcacca tcatcatcac catcatcatc atcatttaga agttttgttt
     5281 caaggtccaa gtagaagagc gagctgcaga ctggctgtgt ataagggagc ctgacattta
     5341 tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt ggctgagatc
     5401 agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg tcatcatgcg
     5461 ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt tatctgacag
     5521 cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact
     5581 ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa ccttaaactg
     5641 catttcacca gcccctgttc tcgtcagcaa aagagccgtt catttcaata aaccgggcga
     5701 cctcagccat cccttcctga ttttccgctt tccagcgttc ggcacgcaga cgacgggctt
     5761 cattctgcat ggttgtgctt accagaccgg agatattgac atcatatatg ccttgagcaa
     5821 ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc ttcatagcat acctcttttt
     5881 gacatacttc gggtatacat atcagtatat attcttatac cgcaaaaatc agcgcgcaaa
     5941 tacgcatact gttatctggc ttttagtaag ccggatccac gcgtttacgc cccgccctgc
     6001 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa
     6061 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
     6121 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa
     6181 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
     6241 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac
     6301 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg
     6361 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc
     6421 atacggaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa
     6481 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc
     6541 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
     6601 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct
     6661 cctgaaaatc tcgacggatc ctaactcaaa atccacacat tatacgagcc ggaagcataa
     6721 agtgtaaagc ctggggtgcc taatgcggcc gccatagtga ctggatatgt tgtgttttac
     6781 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat
     6841 tttacgtttc tcgttcagct tttttgtaca aacttgtgat atcgaattcg ctagctacct
     6901 ttctcttctt ttttggatct acctttctct tcttttttgg ggatccacta gttcagctct
     6961 tctgcataat aactac
//
