LOCUS       sequence-425650-        4124 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 4124)
  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 4124)
  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-15a
FEATURES             Location/Qualifiers
     source          1..4124
                     /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(1051..1053,1054..1767)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1093..1114)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1354..1373)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1701..1722
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1845..1874
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1890..1907
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1986..2232
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2247..2659
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2666..2713
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2732..3106
                     /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      3172..3419
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3188..3206)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3494..4082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3574..3593)
                     /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 tccagggcgc catggccaaa gtgagcaagg gcgaggagct gttcaccggg
     1081 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1141 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1201 ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgcagtgc
     1261 ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1321 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1381 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1441 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1501 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1561 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1621 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1681 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1741 ctcggcatgg acgagctgta caagtaaggt accggatccg aattcgagct ccgtcgacaa
     1801 gcttgcggcc gcactcgagc cgcggcggcc gccagcttgg gcccgaacaa aaactcatct
     1861 cagaagagga tctgaatagc gccgtcgacc atcatcatca tcatcattga gttttagcct
     1921 tagacatgac tgttcctcag ttcaagttgg gcacttacga gaagaccggt cttgctagat
     1981 tctaatcaag aggatgtcag aatgccattt gcctgagaga tgcaggcttc atttttgata
     2041 cttttttatt tgtaacctat atagtatagg attttttttg tcattttgtt tcttctcgta
     2101 cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt aggggtttgg
     2161 gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca gagtacagaa
     2221 gattaagtga gaccttcgtt tgtgcggatc ccccacacac catagcttca aaatgtttct
     2281 actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa
     2341 cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc
     2401 cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa
     2461 aggcaataaa aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc
     2521 tctttcgatg acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt
     2581 cagtttcatt tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag
     2641 catagcaatc taatctaagg gcggtgttga caattaatca tcggcatagt atatcggcat
     2701 agtataatac gacaaggtga ggaactaaaa catggccaag ttgaccagtg ccgttccggt
     2761 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
     2821 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
     2881 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
     2941 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
     3001 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
     3061 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tccgacgcgg
     3121 cccgacgggt ccgaggcctc ggagatccgt cccccttttc ctttgtcgat atcatgtaat
     3181 tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag
     3241 gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa
     3301 gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta
     3361 acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgca
     3421 agctggagac caacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3481 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3541 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3601 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3661 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3721 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3781 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3841 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3901 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3961 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4021 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4081 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtgg
//
