LOCUS       sequence-85572-e        4871 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Pichia pastoris 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 4871)
  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 4871)
  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: pCoofy42
FEATURES             Location/Qualifiers
     source          1..4871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             join(941..997,998..1195,1196..1207)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence. The EA dipeptides are then removed by 
                     dipeptidyl aminopeptidase A."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
     CDS             1219..1242
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             1279..1287
                     /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             1289..1597
                     /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     1476..1495
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1601..1630
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(1634..1639,1640..1663,1664..1699,1700..1723)
                     /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             1727..1771
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1778..1855
                     /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             1860..1895
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             2553..2582
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2598..2615
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2695..2941
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2956..3367
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3375..3422
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3441..3815
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      3881..4128
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3897..3915)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4203..4791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4283..4302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
       61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
      121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
      181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
      241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt
      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
      901 caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt
      961 tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga
     1021 agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga
     1081 tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa
     1141 tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga agagagaggc
     1201 tgaagcatcg atgaattcct ggaagttctg ttccaggggc ccttgacaac ataaaaactt
     1261 tgtgttatac ttgtaacgta aggaggtaat gattcagttt aaggtttaca cctataaaag
     1321 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg
     1381 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     1441 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     1501 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     1561 caaaaacgcc attaacctga tgttctgggg aatataagag catcatcatc atcaccacca
     1621 ccaccaccac tgaagcgctt ggagccaccc gcagttcgaa aaaggtggag gttctggcgg
     1681 tggatcggga ggttcagcgt ggagccaccc gcagttcgag aaatagaaag aaaccgctgc
     1741 tgctaaattc gaacgccagc acatggacag ctagatgaag cggcggtgga agaaaaactt
     1801 tatcgccgtg tccgccgcca accggttcaa gaagatcagc agctctggcg ccctgctgag
     1861 aggaccaggt ggacccccgg ctgatcgacg gcaaatgagg cagcggcaag atcctgcaca
     1921 accagaacgt gaacagctgg ggccccatca ccgtgacccc taccaccgac ggcggcgaga
     1981 caagattcga cggccagatc atcgtgcaga tggaaaacga ccccgtggtg gccaaggccg
     2041 ctgccaatct ggccggcaag cacgccgaga gcagcgtggt ggtgcagctg gacagcgacg
     2101 gcaactaccg ggtggtgtac ggcgacccca gcaagctgga cggcaagctg cggtggcagc
     2161 tggtgggaca cggcagggac cacagcgaga caaacaacac ccggctgagc ggctacagcg
     2221 ccgacgagct ggccgtgaag ctggccaagt tccagcagag cttcaaccag gccgagaaca
     2281 tcaacaacaa gcccgaccac atcagcatcg tgggctgcag cctggtgtcc gacgacaagc
     2341 agaagggctt cggccaccag ttcatcaacg ctatggacgc caacggcctg cgggtggacg
     2401 tgtccgtgcg gtctagcgaa ctggccgtgg acgaggccgg cagaaagcac accaaggacg
     2461 ccaatggcga ctgggtgcag aaggccgaaa acaacaaggt gtccctgagc tgggacgccc
     2521 agggcctgga atgagcggcc gccagctttc tagaacaaaa actcatctca gaagaggatc
     2581 tgaatagcgc cgtcgaccat catcatcatc atcattgagt ttgtagcctt agacatgact
     2641 gttcctcagt tcaagttggg cacttacgag aagaccggtc ttgctagatt ctaatcaaga
     2701 ggatgtcaga atgccatttg cctgagagat gcaggcttca tttttgatac ttttttattt
     2761 gtaacctata tagtatagga ttttttttgt cattttgttt cttctcgtac gagcttgctc
     2821 ctgatcagcc tatctcgcag ctgatgaata tcttgtggta ggggtttggg aaaatcattc
     2881 gagtttgatg tttttcttgg tatttcccac tcctcttcag agtacagaag attaagtgag
     2941 accttcgttt gtgcggatcc cccacacacc atagcttcaa aatgtttcta ctcctttttt
     3001 actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac
     3061 agcatactaa attttccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg
     3121 tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa
     3181 atttttatca cgtttctttt tcttgaaatt ttttttttta gtttttttct ctttcagtga
     3241 cctccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt
     3301 tcttgttcta ttacaacttt ttttacttct tgttcattag aaagaaagca tagcaatcta
     3361 atctaagggg cggtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     3421 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
     3481 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
     3541 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
     3601 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
     3661 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
     3721 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
     3781 actgcgtgca cttcgtggcc gaggagcagg actgacacgt ccgacggcgg cccacgggtc
     3841 ccaggcctcg gagatccgtc ccccttttcc tttgtcgata tcatgtaatt agttatgtca
     3901 cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa
     3961 cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt
     4021 atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg
     4081 aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctggagacc
     4141 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4201 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4261 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4321 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4381 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4441 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4501 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4561 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4621 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     4681 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4741 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4801 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4861 gtcatgagat c
//
