LOCUS       sequence-448286-        5196 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 5196)
  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 5196)
  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-12a
FEATURES             Location/Qualifiers
     source          1..5196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    13..34
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(49..54,55..72,73..79)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    87..103
                     /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)."
     primer_bind     92..114
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     111..127
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     111..127
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     sig_peptide     163..225
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="MKKTAIAIAVALAGFATVAQA"
     CDS             join(235..237,238..951)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(277..298)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(538..557)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     885..906
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1004..1021
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1046..1753
                     /codon_start=1
                     /gene="dsbC"
                     /product="bacterial periplasmic protein disulfide 
                     isomerase"
                     /label=DsbC
                     /translation="MKKGFMLFTLLAAFSGFAQADDAAIQQTLAKMGIKSSDIQPAPVA
                     GMKTVLTNSGVLYITDDGKHIIQGPMYDVSGTAPVNVTNKMLLKQLNALEKEMIVYKAP
                     QEKHVITVFTDITCGYCHKLHEQMADYNALGITVRYLAFPRQGLDSDAEKEMKAIWCAK
                     DKNKAFDDVMAGKSVAPASCDVDIADHYALGVQLGVSGTPAVVLSNGTLVPGYQPPKEM
                     KEFLDEHQKMTSGK"
     rep_origin      1765..2220
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1852..1871)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2062..2083
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2247..2351
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2352..2420,2421..3212)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2570..2589)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3383..3971
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3872..3891
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4036..4113
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4114..5196
                     /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     complement(4133..4152)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
       61 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      121 ctatgaccat gattacgaat ttctagataa cgagggcaaa aaatgaaaaa gacagctatc
      181 gcgattgcag tggcactggc cggcttcgct accgtagcgc aggccatggc caaagtgagc
      241 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
      301 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
      361 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
      421 accttcggct acggcctgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
      481 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
      541 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
      601 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
      661 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
      721 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
      781 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
      841 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
      901 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aggtaccgga
      961 tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcatcatc atcatcatca
     1021 ctgactgaaa agaacgggaa gatttatgaa gaaaggtttt atgttgttta ctttgttagc
     1081 ggcgttttca ggctttgctc aggctgatga cgcggcaatt caacaaacgt tagccaaaat
     1141 gggcatcaaa agcagcgata ttcagcccgc gcctgtagct ggcatgaaga cagttctgac
     1201 taacagcggc gtgttgtaca tcaccgatga tggtaaacat atcattcagg ggccaatgta
     1261 tgacgttagt ggcacggctc cggtcaatgt caccaataag atgctgttaa agcagttgaa
     1321 tgcgcttgaa aaagagatga tcgtttataa agcgccgcag gaaaaacacg tcatcaccgt
     1381 gtttactgat attacctgtg gttactgcca caaactgcat gagcaaatgg cagactacaa
     1441 cgcgctgggg atcaccgtgc gttatcttgc tttcccgcgc caggggctgg acagcgatgc
     1501 agagaaagaa atgaaagcta tctggtgtgc gaaagataaa aacaaagcgt ttgatgatgt
     1561 gatggcaggt aaaagcgtcg caccagccag ttgcgacgtg gatattgccg accattacgc
     1621 acttggcgtc cagcttggcg ttagcggtac tccggcagtt gtgctgagca atggcacact
     1681 tgttccgggt taccagccgc cgaaagagat gaaagaattc ctcgacgaac accaaaaaat
     1741 gaccagcggt aaataaggat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1801 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1861 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1921 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1981 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2041 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2101 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2161 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2221 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2281 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2341 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2401 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2461 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2521 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2581 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2641 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2701 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2761 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2821 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2881 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2941 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3001 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3061 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3121 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3181 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3241 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3301 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3361 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3421 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3481 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     3541 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3601 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3661 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3721 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3781 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3841 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3901 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3961 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4021 ttgctcacat gacccgacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     4081 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     4141 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     4201 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     4261 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     4321 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     4381 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     4441 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     4501 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     4561 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     4621 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     4681 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     4741 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     4801 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     4861 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     4921 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     4981 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     5041 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     5101 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5161 gcacgacagg tttcccgact ggaaagcggg cagtga
//
