LOCUS       sequence-85570-e        5797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for parallel SLIC cloning. C-terminal 
            tags can be added during SLIC..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5797)
  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 5797)
  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: pCoofy47
FEATURES             Location/Qualifiers
     source          1..5797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        42..421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        422..625
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     576..596
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     622..646
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1127..1146
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1304..1327
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1364..1372
                     /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             1374..1682
                     /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     1561..1580
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1686..1715
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(1719..1724,1725..1748,1749..1784,1785..1808)
                     /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             1812..1856
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1863..1940
                     /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             1947..1982
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     polyA_signal    2673..2728
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2727..2746)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3811..3829)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3897..4001
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4002..4070,4071..4862)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4220..4239)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5033..5621
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5522..5541
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5775..5792
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtacatttat attggctcat gtccaatatg accgccatgt tgacattgat tattgactag
       61 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      121 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      181 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      241 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      301 tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      361 gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      421 ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt
      481 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      541 ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg
      601 gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcctca ctctcttccg
      661 catcgctgtc tgcgagggcc agctgttggg ctcgcggttg aggacaaact cttcgcggtc
      721 tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg
      781 acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg tctaaccagt
      841 cacagtcgca aggtaggctg agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt
      901 ttctggcgga ggtgctgctg atgatgtaat taaagtaggc ggtcttgaga cggcggatgg
      961 tcgaggtgag gtgtggcagg cttgagatcc agctgttggg gtgagtactc cctctcaaaa
     1021 gcgggcatta cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc
     1081 acctggcccg atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc
     1141 acaggtgtcc actcccaggt ccaagtttaa acggatctct agcgaattca tgaactttct
     1201 gctgtcctgg gtgcactggt ccctggccct gctgctgtac ctgcaccacg ccaagtggtc
     1261 ccaggccgct cctatggccg agggcggtgg ccagaacgaa ttcctggaag ttctgttcca
     1321 ggggcccttg acaacataaa aactttgtgt tatacttgta acgtaaggag gtaatgattc
     1381 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga
     1441 gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc
     1501 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
     1561 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
     1621 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
     1681 aagagcatca tcatcatcac caccaccacc accactgaag cgcttggagc cacccgcagt
     1741 tcgaaaaagg tggaggttct ggcggtggat cgggaggttc agcgtggagc cacccgcagt
     1801 tcgagaaata gaaagaaacc gctgctgcta aattcgaacg ccagcacatg gacagctaga
     1861 tgaagcggcg gtggaagaaa aactttatcg ccgtgtccgc cgccaaccgg ttcaagaaga
     1921 tcagcagctc tggcgccctg ctgtgagagg accaggtgga cccccggctg atcgatggca
     1981 aatgaggcag cggcaagatc ctgcacaacc agaacgtgaa cagctggggc cccatcaccg
     2041 tgacccctac caccgacggc ggcgagacaa gattcgacgg ccagatcatc gtgcagatgg
     2101 aaaacgaccc cgtggtggcc aaggccgctg ccaatctggc cggcaagcac gccgagagca
     2161 gcgtggtggt gcagctggac agcgacggca actaccgggt ggtgtacggc gaccccagca
     2221 agctggacgg caagctgcgg tggcagctgg tgggacacgg cagggaccac agcgagacaa
     2281 acaacacccg gctgagcggc tacagcgccg acgagctggc cgtgaagctg gccaagttcc
     2341 agcagagctt caaccaggcc gagaacatca acaacaagcc cgaccacatc agcatcgtgg
     2401 gctgcagcct ggtgtccgac gacaagcaga agggcttcgg ccaccagttc atcaacgcta
     2461 tggacgccaa cggcctgcgg gtggacgtgt ccgtgcggtc tagcgaactg gccgtggacg
     2521 aggccggcag aaagcacacc aaggacgcca atggcgactg ggtgcagaag gccgaaaaca
     2581 acaaggtgtc cctgagctgg gacgcccagg gcctggaatg agcggccgct cgaggccggc
     2641 aaggccggat cccccgacct cgacctctgg ctaataaagg aaatttattt tcattgcaat
     2701 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     2761 ggtcgagatc cctcggagat ctctagctag aggatcgatc cccgccccgg acgaactaaa
     2821 cctgactacg acatctctgc cccttcttcg cggggcagtg catgtaatcc cttcagttgg
     2881 ttggtacaac ttgccaactg aaccctaaac gggtagcata tgcttcccgg gtagtagtat
     2941 atactatcca gactaaccct aattcaatag catatgttac ccaacgggaa gcatatgcta
     3001 tcgaattagg gttagtaaaa gggtcctaag gaacagcgat gtaggtgggc gggccaagat
     3061 aggggcgcga ttgctgcgat ctggaggaca aattacacac acttgcgcct gagcgccaag
     3121 cacagggttg ttggtcctca tattcacgag gtcgctgaga gcacggtggg ctaatgttgc
     3181 catgggtagc atatactacc caaatatctg gatagcatat gctatcctaa tctatatctg
     3241 ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa tctatatctg
     3301 ggtagtatat gctatcctaa tttatatctg ggtagcatag gctatcctaa tctatatctg
     3361 ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa tctgtatccg
     3421 ggtagcatat gctatcctaa tagagattag ggtagtatat gctatcctaa tttatatctg
     3481 ggtagcatat actacccaaa tatctggata gcatatgcta tcctaatcta tatctgggta
     3541 gcatatgcta tcctaatcta tatctgggta gcataggcta tcctaatcta tatctgggta
     3601 gcatatgcta tcctaatcta tatctgggta gtatatgcta tcctaattta tatctgggta
     3661 gcataggcta tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta
     3721 gtatatgcta tcctaatctg tatccgggta gcatatgcta tcctcatgat aagctgtcaa
     3781 acatgagaat taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     3841 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     3901 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3961 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4021 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4081 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4141 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4201 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     4261 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4321 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4381 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4441 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4501 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     4561 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4621 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4681 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4741 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4801 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4861 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4921 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4981 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5041 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5101 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5161 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5221 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5281 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5341 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5401 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5461 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5521 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5581 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5641 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5701 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5761 gaacgaccga gcgcagcgag tcagtgagcg aggaagc
//
