LOCUS       Exported                5828 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression of bovine pancreatic ribonuclease A.
ACCESSION   .
VERSION     .
KEYWORDS    pET22b RNase A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5828)
  AUTHORS   delCardayre SB, Ribo M, Yokel EM, Quirk DJ, Rutter WJ, Raines RT
  TITLE     Engineering ribonuclease A: production, purification and 
            characterization of wild-type enzyme and mutants at Gln11.
  JOURNAL   Protein Eng. 1995 Mar;8(3):261-73.
  PUBMED    7479688
REFERENCE   2  (bases 1 to 5828)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5828
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     7..26
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     110..129
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        110..128
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    129..153
                     /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             168..190
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     198..263
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
     CDS             264..308
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             668..685
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(738..756)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      752..799
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      836..1291
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(923..942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1133..1154
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1317..1421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1422..2282
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1640..1659)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2453..3041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2942..2961
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3195..3212
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3227..3366
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3292..3311)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3452..3474
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3468..3659)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5513..5532
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5552..5629)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
       61 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
      121 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
      181 aagaaggaga tatacatatg aaatacctgc tgccgaccgc tgctgctggt ctgctgctcc
      241 tcgctgccca gccggcgatg gccaaggaaa ctgcagcagc caagtttgag cggcagcaca
      301 tggactccag cacttccgct gccagcagct ccaactactg taaccagatg atgaagagcc
      361 ggaacctgac caaagatcga tgcaagccag tgaacacctt tgtgcacgag tccctggctg
      421 atgtccaggc cgtgtgctcc cagaaaaatg ttgcctgcaa gaatgggcag accaattgct
      481 accagagcta ctccaccatg agcatcaccg actgccgtga gaccggcagc tccaagtacc
      541 ccaactgtgc ctacaagacc acccaggcga ataaacacat cattgtggct tgtgagggaa
      601 acccgtacgt gccagtccac tttgatgctt cagtgtagta gtcgacaagc ttgcggccgc
      661 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
      721 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      781 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
      841 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      901 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      961 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1021 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1081 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     1141 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1201 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1261 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     1321 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1381 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1441 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1501 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1561 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1621 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1681 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1741 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1801 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1861 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1921 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gccagctgca atggcaacaa
     1981 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2041 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2101 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2161 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2221 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2281 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2341 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2401 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2461 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2521 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2581 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2641 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2701 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2761 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2821 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2881 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2941 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3001 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3061 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3121 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3181 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3241 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3301 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3361 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3421 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3481 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3541 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3601 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3661 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3721 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3781 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3841 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3901 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3961 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4021 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4081 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4141 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4201 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4261 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4321 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4381 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4441 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4501 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4561 cagggtggtt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     4621 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     4681 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     4741 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4801 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     4861 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     4921 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     4981 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     5041 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     5101 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     5161 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     5221 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     5281 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     5341 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     5401 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     5461 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     5521 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     5581 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     5641 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     5701 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     5761 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     5821 cgatcttc
//
