LOCUS       sequence-170797-        6441 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 6441)
  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 6441)
  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-13a
FEATURES             Location/Qualifiers
     source          1..6441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     161..180
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     264..283
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        264..282
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    283..307
                     /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             322..344
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             364..387
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             760..777
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(799..801,802..1515)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(841..862)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1102..1121)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1449..1470
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1568..1585
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1638..1656)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1652..1699
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1736..2191
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1823..1842)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2033..2054
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2283..3098)
                     /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(2283..3098)
                     /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     3006..3025
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3090..3109
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3220..3808
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3709..3728
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3962..3979
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3994..4133
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4059..4078)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4219..4241
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4235..4426)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6280..6299
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6319..6396)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
       61 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
      121 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
      181 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
      241 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
      301 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgggcagc
      361 agccatcatc atcatcatca tcatcacaac actagtagca attccatgtt ttgtacattt
      421 tttgaaaaac atcaccggaa gtgggacata ctgttagaaa aaagcacggg tgtgatggaa
      481 gctatgaaag tgacgagtga ggaaaaggaa cagctgagca cagcaatcga ccgaatgaat
      541 gaaggactgg acgcgtttat ccagctgtat aatgaatcgg aaattgatga accgcttatt
      601 cagcttgatg atgatacagc cgagttaatg aagcaggccc gagatatgta cggccaggaa
      661 aagctaaatg agaaattaaa tacaattatt aaacagattt tatccatctc agtatctgaa
      721 gaaggcgaaa aagaaggttc tggttctggt tctggttctc tggtgcctcg tggcagcggt
      781 ggaggtgcca tggccaaagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      841 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      901 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      961 gtgccctggc ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt cgcccgctac
     1021 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1081 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1141 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1201 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     1261 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     1321 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1381 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1441 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1501 gagctgtaca agtaaggtac cggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc
     1561 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     1621 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1681 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     1741 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1801 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1861 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1921 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1981 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2041 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2101 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2161 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     2221 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2281 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2341 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2401 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2461 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2521 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2581 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2641 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2701 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2761 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2821 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2881 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2941 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     3001 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3061 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     3121 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     3181 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3241 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3301 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3361 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3421 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3481 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3541 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3601 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3661 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3721 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3781 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3841 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3901 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3961 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4021 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     4081 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     4141 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4201 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4261 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4321 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4381 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4441 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4501 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4561 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4621 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4681 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4741 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4801 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4861 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4921 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4981 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     5041 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     5101 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5161 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5221 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5281 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtnggtttt
     5341 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5401 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5461 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     5521 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5581 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5641 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5701 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5761 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5821 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5881 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5941 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     6001 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     6061 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     6121 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     6181 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     6241 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     6301 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     6361 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     6421 gcgactcctg cattaggaag c
//
