LOCUS       sequence-298771-        6994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of an RNase inhibitor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6994)
  TITLE     COVID-19 Diagnostic Toolkit Enzymes
REFERENCE   2  (bases 1 to 6994)
  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"
COMMENT     Sequence Label: pmal_c5x_RNAse_Inhib
FEATURES             Location/Qualifiers
     source          1..6994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(152..157,158..173,174..180)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    188..204
                     /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     193..215
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             274..1374
                     /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
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1348..1371
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1423..1434
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     terminator      2826..2912
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3004..3031
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3059..3149
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3150..3218,3219..4010)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3368..3387)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4098..4686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4587..4606
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4840..4857
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5040..5062
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5056..5247)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5743..5820
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5821..6903
                     /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(5840..5859)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat
       61 aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata
      121 acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg
      181 tgtggaattg tgagcggata acaatttcac acaggaaaca gccagtccgt ttaggtgttt
      241 tcacgagcaa ttgaccaaca aggaccatag attatgaaaa tcgaagaagg taaactggta
      301 atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag
      361 aaagataccg gaattaaagt caccgttgag catccggata aactggaaga gaaattccca
      421 caggttgcgg caactggcga tggccctgac attatcttct gggcacacga ccgctttggt
      481 ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag
      541 ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc
      601 gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc
      661 tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg
      721 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
      781 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
      841 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc
      901 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
      961 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
     1021 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
     1081 gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa
     1141 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
     1201 gaagagttgg tgaaagatcc gcgtattgcc gccactatgg aaaacgccca gaaaggtgaa
     1261 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
     1321 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg
     1381 agctcgaaca acaacaacaa taacaataac aacaacctcg ggattgaggg aaggagtctt
     1441 gacatccagt gtgagcagct gagtgatgcc cggtggacag agctccttcc tctgatccag
     1501 caatacgaag tggtcaggct ggatgactgt ggcctcactg aagtgcggtg caaagacatc
     1561 agctcagcag tccaagccaa ccctgccctg acagagctca gccttcgcac caatgaactg
     1621 ggtgatggcg gtgtgggtct ggtgctccag ggcctgcaga atcccacttg taagatccag
     1681 aagctgagcc ttcagaactg cggcttgacg gaggctggct gtgggatcct gcctggcatg
     1741 ctgcgctctt tgtctaccct gcgtgagcta catctcaatg ataaccctat gggggacgca
     1801 ggcctgaagc tgctctgtga aggacttcag gacccccagt gccgtcttga gaagcttcag
     1861 ttggaatact gtaacctcac agctaccagc tgcgagcccc tggcttcagt gctcagggtg
     1921 aaagctgact ttaaggagct agtattgagc aacaacgacc tccatgaacc tggtgtccgt
     1981 attctgtgcc agggcctgaa ggactctgcc tgtcaactgg agtcactcaa gctggagaac
     2041 tgtggtatca cagcagccaa ctgcaaggat ctgtgcgatg ttgtggcctc caaagcttcg
     2101 ctacaggaac tggacttgag cagcaacaag ctaggtaatg caggcattgc agcactgtgc
     2161 ccaggactgc tgcttcccag ctgtaagctc aggactctgt ggctctggga atgtgatatc
     2221 actgcagagg gctgcaagga cctgtgccgt gtcctcagag ccaagcagag cctgaaggaa
     2281 ctcagccttg cttccaatga gctgaaggat gagggtgccc ggctgctgtg tgagagccta
     2341 ttagagcctg gctgtcagct ggagtcactg tggataaaga cctgtagcct cacagctgcc
     2401 tcctgtccct acttctgctc cgtgttgacc aaaagccgtt ctctgttgga gctgcaaatg
     2461 agcagcaacc cactgggaga cgagggagtc caggagcttt gcaaggccct gagccagcca
     2521 gatacagtgc tgcgtgagct ttggttggga gactgtgatg tgacaaatag tggttgcagc
     2581 agccttgcca atgtcctgct ggccaaccgc agcctgaggg aactggacct cagtaacaac
     2641 tgcatggggg gcccaggtgt cctgcaactg ctggagagcc tcaaacagcc cagctgcacc
     2701 cttcagcagc tcgtcctgta tgacatttac tggacaaatg aggtggaaga acagcttcgg
     2761 gccctggagg aggaaaggcc ttccctgagg atcatttcct aacctgcagg taattaaata
     2821 agcttcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     2881 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga
     2941 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta
     3001 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt tcggtccgtt
     3061 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3121 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3181 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3241 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3301 gcggtaagat ccttgagagt tttcgccccg aagaacgttt cccaatgatg agcactttta
     3361 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     3421 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3481 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3541 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3601 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3661 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3721 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3781 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3841 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3901 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3961 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4021 aagtttactc atatatactt tagattgatt tccttaggac tgagcgtcaa ccccgtagaa
     4081 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4141 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4201 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4261 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4321 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4381 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4441 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4501 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4561 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4621 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4681 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4741 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4801 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4861 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4921 atataaggtg cactgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4981 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5041 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5101 gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc
     5161 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5221 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5281 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5341 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5401 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5461 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5521 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5581 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5641 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5701 catgcgcacc cgtggccagg acccaacgct gcccgaaatt ccgacaccat cgaatggtgc
     5761 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     5821 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     5881 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     5941 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     6001 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     6061 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     6121 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     6181 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     6241 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     6301 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     6361 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     6421 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     6481 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     6541 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     6601 gggctgcgcg ttggtgcgga tatttcggta gtgggatacg acgataccga agacagctca
     6661 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     6721 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     6781 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cacaaaccgc ctctccccgc
     6841 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     6901 tgagcgcaac gcaattaatg taagttagct cactcattag gcacaattct catgtttgac
     6961 agcttatcat cgactgcacg gtgcaccaat gctt
//
