LOCUS       sequence-464535-        7262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector for parallel SLIC cloning containing 
            N-terminal 6x His and GST tags. C-terminal tags can be added during 
            SLIC..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7262)
  AUTHORS   Scholz J, Besir H, Strasser C, Suppmann S
  TITLE     A new method to customize protein expression vectors for fast, 
            efficient and background free parallel cloning.
  JOURNAL   BMC Biotechnol. 2013 Feb 14;13(1):12.
  PUBMED    23410102
REFERENCE   2  (bases 1 to 7262)
  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: "BMC 
            Biotechnol."; date: "2013-02-14"; volume: "13(1)"; pages: "12"
COMMENT     Sequence Label: pCoofy48
FEATURES             Location/Qualifiers
     source          1..7262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(559..1374)
                     /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(559..1374)
                     /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     1282..1301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1366..1385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1496..2084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1985..2004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2238..2255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2270..2409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2335..2354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2495..2517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2511..2702)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3511..4593)
                     /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     4555..4574
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4594..4671)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4877..4896
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4980..4999
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4980..4998
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4999..5023
                     /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             5038..5060
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5073..5090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5109..5762
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             5769..5792
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             5833..5841
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             5843..6151
                     /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="MIQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSD
                     KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     6030..6049
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             6155..6184
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(6188..6193,6194..6217,6218..6253,6254..6277)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             6281..6325
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             6332..6409
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             6416..6451
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             7106..7123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7176..7194)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7190..7237
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     2581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     2881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     2941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     3181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     3241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     3301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     3361 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     3421 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3481 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3601 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3661 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3721 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3781 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3841 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     3901 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3961 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4021 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4081 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4141 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4201 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4261 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4321 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4381 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4441 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4501 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4561 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4621 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4681 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     4741 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     4801 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     4861 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     4921 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     4981 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     5041 gtttaacttt aagaaggaga tataccatga aacatcacca tcaccatcac aacactagta
     5101 gcaattccat gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc
     5161 gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag
     5221 gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt
     5281 atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca
     5341 agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag
     5401 cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc
     5461 tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat
     5521 gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg
     5581 ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt
     5641 gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt
     5701 atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa
     5761 aatcggatct ggaagttctg ttccaggggc ccatggttga caacataaaa actttgtgtt
     5821 atacttgtaa cgtaaggagg taatgattca gtttaaggtt tacacctata aaagagagag
     5881 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat
     5941 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
     6001 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
     6061 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
     6121 cgccattaac ctgatgttct ggggaatata agagcatcat catcatcacc accaccacca
     6181 ccactgaagc gcttggagcc acccgcagtt cgaaaaaggt ggaggttctg gcggtggatc
     6241 gggaggttca gcgtggagcc acccgcagtt cgagaaatag aaagaaaccg ctgctgctaa
     6301 attcgaacgc cagcacatgg acagctagat gaagcggcgg tggaagaaaa actttatcgc
     6361 cgtgtccgcc gccaaccggt tcaagaagat cagcagctct ggcgccctgc tgtgagagga
     6421 ccaggtggac ccccggctga tcgatggcaa atgaggcagc ggcaagatcc tgcacaacca
     6481 gaacgtgaac agctggggcc ccatcaccgt gacccctacc accgacggcg gcgagacaag
     6541 attcgacggc cagatcatcg tgcagatgga aaacgacccc gtggtggcca aggccgctgc
     6601 caatctggcc ggcaagcacg ccgagagcag cgtggtggtg cagctggaca gcgacggcaa
     6661 ctaccgggtg gtgtacggcg accccagcaa gctggacggc aagctgcggt ggcagctggt
     6721 gggacacggc agggaccaca gcgagacaaa caacacccgg ctgagcggct acagcgccga
     6781 cgagctggcc gtgaagctgg ccaagttcca gcagagcttc aaccaggccg agaacatcaa
     6841 caacaagccc gaccacatca gcatcgtggg ctgcagcctg gtgtccgacg acaagcagaa
     6901 gggcttcggc caccagttca tcaacgctat ggacgccaac ggcctgcggg tggacgtgtc
     6961 cgtgcggtct agcgaactgg ccgtggacga ggccggcaga aagcacacca aggacgccaa
     7021 tggcgactgg gtgcagaagg ccgaaaacaa caaggtgtcc ctgagctggg acgcccaggg
     7081 cctggaatga gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     7141 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     7201 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     7261 at
//
