LOCUS       sequence-249042-        5913 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E.coli vector for parallel SLIC cloning containing N-terminal 
            His6-3xAvi with PreScission Cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5913)
  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 5913)
  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: pCoofy52
FEATURES             Location/Qualifiers
     source          1..5913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    16..40
                     /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             55..77
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             90..107
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             123..167
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             183..227
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             243..287
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             297..320
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             436..444
                     /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             446..754
                     /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     633..652
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             774..791
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(844..862)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      858..905
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      942..1397
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1029..1048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1239..1260
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1489..2304)
                     /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(1489..2304)
                     /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     2212..2231
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2296..2315
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2426..3014
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2915..2934
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3168..3185
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3200..3339
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3265..3284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3425..3447
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3441..3632)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4441..5523)
                     /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     5485..5504
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5524..5601)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5807..5826
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5910..5913,1..16)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5910..5913,1..15)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
       61 taactttaag aaggagatat accatgaaac atcaccatca ccatcactcc gcggatatgt
      121 ccggcctgaa cgacatcttc gaggctcaga aaatcgaatg gcacgaatcc gcggatatgt
      181 ccggcctgaa cgacatcttc gaggctcaga aaatcgaatg gcacgaatcc gcggatatgt
      241 ccggcctgaa cgacatcttc gaggctcaga aaatcgaatg gcacgaatcc gcgggtctgg
      301 aagttctgtt ccaggggccc atggactaat gaggtaccgg atccgaattc gagctccgtc
      361 gacaagcttg atatcgaatt cctgcagccc aaactgcagt tgacaacata aaaactttgt
      421 gttatacttg taacgtaagg aggtaatgat tcagtttaag gtttacacct ataaaagaga
      481 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg
      541 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
      601 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
      661 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
      721 aaacgccatt aacctgatgt tctggggaat ataagcttgc ggccgcactc gagcaccacc
      781 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
      841 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
      901 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
      961 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1021 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1081 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1141 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1201 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1261 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1321 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1381 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1441 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     1501 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     1561 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     1621 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     1681 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     1741 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     1801 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     1861 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     1921 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     1981 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     2041 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     2101 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     2161 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     2221 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     2281 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     2341 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2401 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2461 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2521 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2581 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2641 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2701 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2761 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2821 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2881 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2941 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3001 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3061 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3121 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3181 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3241 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3301 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3361 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3421 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3481 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3541 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3601 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3661 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3721 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3781 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3841 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     3901 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     3961 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     4021 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4081 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     4141 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     4201 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     4261 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     4321 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     4381 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4441 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4501 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4561 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4621 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4681 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     4741 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4801 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     4861 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     4921 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     4981 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5041 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5101 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5161 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     5221 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     5281 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     5341 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     5401 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5461 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5521 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5581 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5641 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     5701 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     5761 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     5821 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     5881 agaggatcga gatctcgatc ccgcgaaatt aat
//
