LOCUS       sequence-442937-        7177 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 7177)
  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 7177)
  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-1a
FEATURES             Location/Qualifiers
     source          1..7177
                     /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             94..111
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             115..1215
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1189..1212
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1240..1260
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1273..1275,1276..1989)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1315..1336)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1576..1595)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1923..1944
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2042..2059
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2112..2130)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2126..2173
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2210..2665
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2297..2316)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2507..2528
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2757..3572)
                     /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(2757..3572)
                     /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     3480..3499
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3564..3583
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3694..4282
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4183..4202
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4436..4453
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4468..4607
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4533..4552)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4693..4715
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4709..4900)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5709..6791)
                     /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     6753..6772
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6792..6869)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7075..7094
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg aaacatcacc atcaccatca ccccatgaaa
      121 atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa cggtctcgct
      181 gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga gcatccggat
      241 aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga cattatcttc
      301 tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga aatcaccccg
      361 gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg ttacaacggc
      421 aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa caaagatctg
      481 ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga actgaaagcg
      541 aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg gccgctgatt
      601 gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat taaagacgtg
      661 ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct gattaaaaac
      721 aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa taaaggcgaa
      781 acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag caaagtgaat
      841 tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg
      901 ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa
      961 aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc gctgggtgcc
     1021 gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc cgccactatg
     1081 gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc tttctggtat
     1141 gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga tgaagccctg
     1201 aaagacgcgc agactaattc gggatctggc agtggttctg agaatcttta ttttcagggc
     1261 gccatggcca aagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1321 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1381 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1441 tggcccaccc tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac
     1501 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1561 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1621 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1681 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1741 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1801 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1861 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1921 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1981 tacaagtaag gtaccggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga
     2041 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     2101 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     2161 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     2221 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2281 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2341 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2401 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2461 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2521 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2581 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     2641 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2701 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     2761 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     2821 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     2881 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     2941 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     3001 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     3061 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     3121 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     3181 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     3241 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     3301 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     3361 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     3421 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     3481 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     3541 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     3601 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3661 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3721 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3781 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3841 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3901 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3961 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4021 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4081 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4141 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4201 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4261 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4321 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4381 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4441 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     4501 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4561 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     4621 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     4681 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4741 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     4801 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     4861 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     4921 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     4981 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     5041 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     5101 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     5161 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     5221 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     5281 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     5341 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     5401 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     5461 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     5521 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     5581 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     5641 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     5701 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5761 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     5821 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     5881 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     5941 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     6001 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     6061 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     6121 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     6181 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     6241 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     6301 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     6361 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     6421 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     6481 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     6541 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     6601 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     6661 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     6721 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     6781 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     6841 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     6901 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6961 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     7021 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     7081 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     7141 tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
