LOCUS       sequence-425651-        4128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4128)
  AUTHORS   Goulas T, Cuppari A, Garcia-Castellanos R, Snipas S, Glockshuber R, 
            Arolas JL, Gomis-Ruth FX
  TITLE     The pCri System: A Vector Collection for Recombinant Protein 
            Expression and Purification.
  JOURNAL   PLoS One. 2014 Nov 11;9(11):e112643. doi: 
            10.1371/journal.pone.0112643. eCollection 2014.
  PUBMED    25386923
REFERENCE   2  (bases 1 to 4128)
  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: "PLoS One.";
            date: "2014-11-11"; volume: "9(11):e112643. doi"; pages: " 
            10.1371/journal.pone.0112643. eCollection 2014"
COMMENT     Sequence Label: pCri-15b
FEATURES             Location/Qualifiers
     source          1..4128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..978
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             991..1008
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1018..1038
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1054..1056,1057..1770)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1096..1117)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1357..1376)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1704..1725
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1848..1877
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1893..1910
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1989..2235
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2250..2663
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2670..2717
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2736..3110
                     /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      3176..3423
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3192..3210)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3498..4086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3578..3597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgaaaact cacgttaagg gattttggtc atgagatcag atctaacatc caaagacgaa
       61 aggttgaatg aaaccttttt gccatccgac atccacaggt ccattctcac acataagtgc
      121 caaacgcaac aggaggggat acactagcag cagaccgttg caaacgcagg acctccactc
      181 ctcttctcct caacacccac ttttgccatc gaaaaaccag cccagttatt gggcttgatt
      241 ggagctcgct cattccaatt ccttctatta ggctactaac accatgactt tattagcctg
      301 tctatcctgg cccccctggc gaggttcatg tttgtttatt tccgaatgca acaagctccg
      361 cattacaccc gaacatcact ccagatgagg gctttctgag tgtggggtca aatagtttca
      421 tgttccccaa atggcccaaa actgacagtt taaacgctgt cttggaacct aatatgacaa
      481 aagcgtgatc tcatccaaga tgaactaagt ttggttcgtt gaaatgctaa cggccagttg
      541 gtcaaaaaga aacttccaaa agtcggcata ccgtttgtct tgtttggtat tgattgacga
      601 atgctcaaaa ataatctcat taatgcttag cgcagtctct ctatcgcttc tgaaccccgg
      661 tgcacctgtg ccgaaacgca aatggggaaa cacccgcttt ttggatgatt atgcattgtc
      721 tccacattgt atgcttccaa gattctggtg ggaatactgc tgatagccta acgttcatga
      781 tcaaaattta actgttctaa cccctacttg acagcaatat ataaacagaa ggaagctgcc
      841 ctgtcttaaa cctttttttt tatcatcatt attagcttac tttcataatt gcgactggtt
      901 ccaattgaca agcttttgat tttaacgact tttaacgaca acttgagaag atcaaaaaac
      961 aactaattat tcgaaacgat gggcagcagc catcatcatc atcatcacag cagcggcgaa
     1021 aacctttact tccagggcca tatggctagc aaagtgagca agggcgagga gctgttcacc
     1081 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1141 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1201 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgcag
     1261 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1321 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1381 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1441 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1501 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1561 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1621 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1681 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1741 actctcggca tggacgagct gtacaagtaa ggtaccggat ccgaattcga gctccgtcga
     1801 caagcttgcg gccgcactcg agccgcggcg gccgccagct tgggcccgaa caaaaactca
     1861 tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagttttag
     1921 ccttagacat gactgttcct cagttcaagt tgggcactta cgagaagacc ggtcttgcta
     1981 gattctaatc aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg
     2041 atactttttt atttgtaacc tatatagtat aggatttttt ttgtcatttt gtttcttctc
     2101 gtacgagctt gctcctgatc agcctatctc gcagctgatg aatatcttgt ggtaggggtt
     2161 tgggaaaatc attcgagttt gatgtttttc ttggtatttc ccactcctct tcagagtaca
     2221 gaagattaag tgagaccttc gtttgtgcgg atcccccaca caccatagct tcaaaatgtt
     2281 tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca
     2341 aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt gtcgttaatt
     2401 acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga
     2461 aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt ttttgatttt
     2521 tttctctttc gatgacctcc cattgatatt taagttaata aacggtcttc aatttctcaa
     2581 gtttcagttt catttttctt gttctattac aacttttttt acttcttgct cattagaaag
     2641 aaagcatagc aatctaatct aagggcggtg ttgacaatta atcatcggca tagtatatcg
     2701 gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc
     2761 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
     2821 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
     2881 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
     2941 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
     3001 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
     3061 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtccgac
     3121 gcggcccgac gggtccgagg cctcggagat ccgtccccct tttcctttgt cgatatcatg
     3181 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag
     3241 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta
     3301 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca
     3361 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt
     3421 tgcaagctgg agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3481 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3661 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3901 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtgg
//
