LOCUS       sequence-244909-        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        52..431
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        432..635
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     586..606
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     632..656
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1137..1156
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1314..1337
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1374..1382
                     /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             1384..1692
                     /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     1571..1590
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1696..1725
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(1729..1734,1735..1758,1759..1794,1795..1818)
                     /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             1822..1866
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1873..1950
                     /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             1957..1992
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     polyA_signal    2683..2738
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2737..2756)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3821..3839)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3907..4011
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4012..4080,4081..4872)
                     /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(4230..4249)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5043..5631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5532..5551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(5785..5797,1..5)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgaggaagc gtacatttat attggctcat gtccaatatg accgccatgt tgacattgat
       61 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      121 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      181 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      241 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      301 atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      361 cccagtacat gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg
      421 ctattaccat ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact
      481 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      541 atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta
      601 ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcctca
      661 ctctcttccg catcgctgtc tgcgagggcc agctgttggg ctcgcggttg aggacaaact
      721 cttcgcggtc tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtactccg
      781 ccaccgaggg acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg
      841 tctaaccagt cacagtcgca aggtaggctg agcaccgtgg cgggcggcag cgggtggcgg
      901 tcggggttgt ttctggcgga ggtgctgctg atgatgtaat taaagtaggc ggtcttgaga
      961 cggcggatgg tcgaggtgag gtgtggcagg cttgagatcc agctgttggg gtgagtactc
     1021 cctctcaaaa gcgggcatta cttctgcgct aagattgtca gtttccaaaa acgaggagga
     1081 tttgatattc acctggcccg atctggccat acacttgagt gacaatgaca tccactttgc
     1141 ctttctctcc acaggtgtcc actcccaggt ccaagtttaa acggatctct agcgaattca
     1201 tgaactttct gctgtcctgg gtgcactggt ccctggccct gctgctgtac ctgcaccacg
     1261 ccaagtggtc ccaggccgct cctatggccg agggcggtgg ccagaacgaa ttcctggaag
     1321 ttctgttcca ggggcccttg acaacataaa aactttgtgt tatacttgta acgtaaggag
     1381 gtaatgattc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     1441 gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt
     1501 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
     1561 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
     1621 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     1681 tggggaatat aagagcatca tcatcatcac caccaccacc accactgaag cgcttggagc
     1741 cacccgcagt tcgaaaaagg tggaggttct ggcggtggat cgggaggttc agcgtggagc
     1801 cacccgcagt tcgagaaata gaaagaaacc gctgctgcta aattcgaacg ccagcacatg
     1861 gacagctaga tgaagcggcg gtggaagaaa aactttatcg ccgtgtccgc cgccaaccgg
     1921 ttcaagaaga tcagcagctc tggcgccctg ctgtgagagg accaggtgga cccccggctg
     1981 atcgatggca aatgaggcag cggcaagatc ctgcacaacc agaacgtgaa cagctggggc
     2041 cccatcaccg tgacccctac caccgacggc ggcgagacaa gattcgacgg ccagatcatc
     2101 gtgcagatgg aaaacgaccc cgtggtggcc aaggccgctg ccaatctggc cggcaagcac
     2161 gccgagagca gcgtggtggt gcagctggac agcgacggca actaccgggt ggtgtacggc
     2221 gaccccagca agctggacgg caagctgcgg tggcagctgg tgggacacgg cagggaccac
     2281 agcgagacaa acaacacccg gctgagcggc tacagcgccg acgagctggc cgtgaagctg
     2341 gccaagttcc agcagagctt caaccaggcc gagaacatca acaacaagcc cgaccacatc
     2401 agcatcgtgg gctgcagcct ggtgtccgac gacaagcaga agggcttcgg ccaccagttc
     2461 atcaacgcta tggacgccaa cggcctgcgg gtggacgtgt ccgtgcggtc tagcgaactg
     2521 gccgtggacg aggccggcag aaagcacacc aaggacgcca atggcgactg ggtgcagaag
     2581 gccgaaaaca acaaggtgtc cctgagctgg gacgcccagg gcctggaatg agcggccgct
     2641 cgaggccggc aaggccggat cccccgacct cgacctctgg ctaataaagg aaatttattt
     2701 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     2761 caaatcattt ggtcgagatc cctcggagat ctctagctag aggatcgatc cccgccccgg
     2821 acgaactaaa cctgactacg acatctctgc cccttcttcg cggggcagtg catgtaatcc
     2881 cttcagttgg ttggtacaac ttgccaactg aaccctaaac gggtagcata tgcttcccgg
     2941 gtagtagtat atactatcca gactaaccct aattcaatag catatgttac ccaacgggaa
     3001 gcatatgcta tcgaattagg gttagtaaaa gggtcctaag gaacagcgat gtaggtgggc
     3061 gggccaagat aggggcgcga ttgctgcgat ctggaggaca aattacacac acttgcgcct
     3121 gagcgccaag cacagggttg ttggtcctca tattcacgag gtcgctgaga gcacggtggg
     3181 ctaatgttgc catgggtagc atatactacc caaatatctg gatagcatat gctatcctaa
     3241 tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa
     3301 tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag gctatcctaa
     3361 tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa
     3421 tctgtatccg ggtagcatat gctatcctaa tagagattag ggtagtatat gctatcctaa
     3481 tttatatctg ggtagcatat actacccaaa tatctggata gcatatgcta tcctaatcta
     3541 tatctgggta gcatatgcta tcctaatcta tatctgggta gcataggcta tcctaatcta
     3601 tatctgggta gcatatgcta tcctaatcta tatctgggta gtatatgcta tcctaattta
     3661 tatctgggta gcataggcta tcctaatcta tatctgggta gcatatgcta tcctaatcta
     3721 tatctgggta gtatatgcta tcctaatctg tatccgggta gcatatgcta tcctcatgat
     3781 aagctgtcaa acatgagaat taattcttga agacgaaagg gcctcgtgat acgcctattt
     3841 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     3901 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3961 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4021 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4081 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4141 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4201 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     4261 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4321 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4381 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4441 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4501 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     4561 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4621 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4681 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4741 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4801 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4861 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4921 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4981 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5041 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5101 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5161 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5221 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5281 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5341 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5401 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5461 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5521 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5581 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5641 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5701 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5761 cgccgcagcc gaacgaccga gcgcagcgag tcagtga
//
