LOCUS       sequence-442938-        6001 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 6001)
  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 6001)
  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-7a
FEATURES             Location/Qualifiers
     source          1..6001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(97..99,100..813)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(139..160)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(400..419)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     747..768
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             866..883
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(936..954)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      950..997
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1034..1489
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1121..1140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1331..1352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1581..2396)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1581..2396)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2304..2323
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2388..2407
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2518..3106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3007..3026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3260..3277
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3292..3431
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3357..3376)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3517..3539
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3533..3724)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4533..5615)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5577..5596
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5616..5693)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5899..5918
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gccaaagtga gcaagggcga ggagctgttc
      121 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      181 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      241 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg
      301 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      361 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      421 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
      481 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
      541 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
      601 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
      661 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
      721 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
      781 atcactctcg gcatggacga gctgtacaag taaggtaccg gatccgaatt cgagctccgt
      841 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
      901 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
      961 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1021 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1081 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1141 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1201 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1261 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1321 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1381 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1441 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     1501 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1561 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     1621 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     1681 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     1741 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     1801 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     1861 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     1921 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     1981 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     2041 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     2101 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     2161 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2221 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2281 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     2341 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     2401 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     2461 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2521 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2581 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2641 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     2701 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2761 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2821 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2881 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2941 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3001 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3061 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3121 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3181 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3241 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3301 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     3361 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3421 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3481 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3541 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     3601 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     3661 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     3721 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     3781 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     3841 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     3901 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     3961 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4021 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4081 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4141 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4201 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4261 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4321 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4381 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4441 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4501 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4561 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4621 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     4681 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     4741 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     4801 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     4861 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     4921 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     4981 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5041 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5101 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5161 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5221 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5281 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5341 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5401 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5461 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5521 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5581 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     5641 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     5701 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5761 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     5821 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     5881 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5941 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     6001 t
//
