LOCUS       sequence-243316-        5619 bp    DNA     circular SYN 15-APR-2025
DEFINITION  E.coli vector for parallel SLIC cloning containing N-terminal 
            StrepII with PreScission Cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5619)
  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 5619)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pCoofy9
FEATURES             Location/Qualifiers
     source          1..5619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     71..90
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     178..197
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        178..196
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    197..221
                     /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             236..258
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             274..297
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             304..327
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             427..435
                     /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             437..745
                     /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     624..643
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             768..785
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             792..821
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     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(join(1512..2303,2304..2372))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2135..2154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2373..2465)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2546..3134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3035..3054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3288..3305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3320..3459
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3385..3404)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3545..3567
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3561..3752)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4324..5406)
                     /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     5368..5387
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5407..5484)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
       61 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
      121 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa
      181 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
      241 ttaactttaa gaaggagata taccatggca agctggagcc acccgcagtt cgaaaagggt
      301 gcacttgaag tcctctttca gggacccggg taccaggatc ctgtacaagt cgacaagctt
      361 gatatcgaat tcctgcagcc caaactgcag ttgacaacat aaaaactttg tgttatactt
      421 gtaacgtaag gaggtaatga ttcagtttaa ggtttacacc tataaaagag agagccgtta
      481 tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat
      541 ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt
      601 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
      661 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
      721 taacctgatg ttctggggaa tataagcttg cggccgcaga gctcgctctg gtgccacgcg
      781 gtagttccgc tcatcaccac catcatcacc atcaccacca ctaattaacc taggctgctg
      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 attaacgttt acaatttctg gcggcacgat ggcatgagat tatcaaaaag gatcttcacc
     1441 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     1501 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     1561 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     1621 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     1681 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     1741 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     1801 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     1861 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     1921 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     1981 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2041 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2101 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     2161 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     2221 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     2281 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     2341 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatc atgattgaag
     2401 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2461 acaaataggt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2521 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2581 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2641 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2701 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2761 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2821 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2881 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2941 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3001 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3061 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3121 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3181 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3241 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3301 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3361 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3421 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3481 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3541 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3601 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3661 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3721 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3781 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3841 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3901 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3961 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     4021 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     4081 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     4141 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4201 acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4261 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4321 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4381 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4441 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4501 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4561 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg
     4621 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     4681 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     4741 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     4801 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     4861 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     4921 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     4981 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5041 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5101 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5161 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5221 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5281 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5341 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5401 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5461 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5521 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     5581 catgcaagga gatggcgccc aacagtcccc cggccacgg
//
