LOCUS       sequence-232393-        5827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian vector for parallel SLIC cloning containing the VEGF 
            signal sequence for secretion, N-terminal His, PreScission Cleavage 
            site and different C-tag options.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5827)
  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 5827)
  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: pCoofy57
FEATURES             Location/Qualifiers
     source          1..5827
                     /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="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1334..1357
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1394..1402
                     /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             1404..1712
                     /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     1591..1610
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1716..1745
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(1749..1754,1755..1778,1779..1814,1815..1838)
                     /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             1842..1886
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1893..1970
                     /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             1977..2012
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     polyA_signal    2703..2758
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2757..2776)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3841..3859)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3927..4031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4032..4100,4101..4892)
                     /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(4250..4269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5063..5651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5552..5571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5805..5822
                     /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 ccagaacgct agtcatcacc atcaccatca
     1321 ccatcacgct agcctggaag ttctgttcca ggggcccttg acaacataaa aactttgtgt
     1381 tatacttgta acgtaaggag gtaatgattc agtttaaggt ttacacctat aaaagagaga
     1441 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga
     1501 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc
     1561 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     1621 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     1681 acgccattaa cctgatgttc tggggaatat aagagcatca tcatcatcac caccaccacc
     1741 accactgaag cgcttggagc cacccgcagt tcgaaaaagg tggaggttct ggcggtggat
     1801 cgggaggttc agcgtggagc cacccgcagt tcgagaaata gaaagaaacc gctgctgcta
     1861 aattcgaacg ccagcacatg gacagctaga tgaagcggcg gtggaagaaa aactttatcg
     1921 ccgtgtccgc cgccaaccgg ttcaagaaga tcagcagctc tggcgccctg ctgtgagagg
     1981 accaggtgga cccccggctg atcgacggca aatgaggcag cggcaagatc ctgcacaacc
     2041 agaacgtgaa cagctggggc cccatcaccg tgacccctac caccgacggc ggcgagacaa
     2101 gattcgacgg ccagatcatc gtgcagatgg aaaacgaccc cgtggtggcc aaggccgctg
     2161 ccaatctggc cggcaagcac gccgagagca gcgtggtggt gcagctggac agcgacggca
     2221 actaccgggt ggtgtacggc gaccccagca agctggacgg caagctgcgg tggcagctgg
     2281 tgggacacgg cagggaccac agcgagacaa acaacacccg gctgagcggc tacagcgccg
     2341 acgagctggc cgtgaagctg gccaagttcc agcagagctt caaccaggcc gagaacatca
     2401 acaacaagcc cgaccacatc agcatcgtgg gctgcagcct ggtgtccgac gacaagcaga
     2461 agggcttcgg ccaccagttc atcaacgcta tggacgccaa cggcctgcgg gtggacgtgt
     2521 ccgtgcggtc tagcgaactg gccgtggacg aggccggcag aaagcacacc aaggacgcca
     2581 atggcgactg ggtgcagaag gccgaaaaca acaaggtgtc cctgagctgg gacgcccagg
     2641 gcctggaatg agcggccgct cgaggccggc aaggccggat cccccgacct cgacctctgg
     2701 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact
     2761 cggaaggaca tatgggaggg caaatcattt ggtcgagatc cctcggagat ctctagctag
     2821 aggatcgatc cccgccccgg acgaactaaa cctgactacg acatctctgc cccttcttcg
     2881 cggggcagtg catgtaatcc cttcagttgg ttggtacaac ttgccaactg aaccctaaac
     2941 gggtagcata tgcttcccgg gtagtagtat atactatcca gactaaccct aattcaatag
     3001 catatgttac ccaacgggaa gcatatgcta tcgaattagg gttagtaaaa gggtcctaag
     3061 gaacagcgat gtaggtgggc gggccaagat aggggcgcga ttgctgcgat ctggaggaca
     3121 aattacacac acttgcgcct gagcgccaag cacagggttg ttggtcctca tattcacgag
     3181 gtcgctgaga gcacggtggg ctaatgttgc catgggtagc atatactacc caaatatctg
     3241 gatagcatat gctatcctaa tctatatctg ggtagcatag gctatcctaa tctatatctg
     3301 ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa tttatatctg
     3361 ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa tctatatctg
     3421 ggtagtatat gctatcctaa tctgtatccg ggtagcatat gctatcctaa tagagattag
     3481 ggtagtatat gctatcctaa tttatatctg ggtagcatat actacccaaa tatctggata
     3541 gcatatgcta tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta
     3601 gcataggcta tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta
     3661 gtatatgcta tcctaattta tatctgggta gcataggcta tcctaatcta tatctgggta
     3721 gcatatgcta tcctaatcta tatctgggta gtatatgcta tcctaatctg tatccgggta
     3781 gcatatgcta tcctcatgat aagctgtcaa acatgagaat taattcttga agacgaaagg
     3841 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     3901 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3961 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4021 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4081 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4141 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4201 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4261 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4321 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4381 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4441 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4501 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4561 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4621 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4681 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4741 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     4801 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4861 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4921 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4981 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5041 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5101 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5161 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5221 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5281 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5341 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5401 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5461 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5521 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5581 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5641 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5701 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     5761 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     5821 aggaagc
//
