LOCUS       sequence-482370-        4438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Over-express his(10)-PKA kinase motif-PreScission protease site-Sc 
            RFA3 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4438)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 4438)
  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: Sc hk/P-RFA3-pCDFDuet
FEATURES             Location/Qualifiers
     source          1..4438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     879..898
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(918..995)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1041..1059
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1060..1084
                     /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)."
     CDS             1134..1163
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1176..1187
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1224..1235
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1242..1265
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        1863..1881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1882..1906
                     /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)."
     CDS             2015..2059
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2097..2115)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2111..2158
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2329..3120)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(3121..3212)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(join(4273..4438,1..917))
                     /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"
ORIGIN
        1 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
       61 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
      121 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
      181 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
      241 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
      301 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
      361 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
      421 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      481 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
      541 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      601 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      661 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      721 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      781 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      841 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      901 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
      961 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     1021 cgactcctgc attaggaaat taatacgact cactataggg gaattgtgag cggataacaa
     1081 ttcccctgta gaaataattt tgtttaactt taataaggag atataccatg agccatcatc
     1141 atcatcatca tcatcatcat cacagcagcg gccatatcga aggtcgtcat attctgcgcc
     1201 gcgcgagcgt gagcagcggc catattgaag gccgccatat tctggaagtt ctgttccagg
     1261 ggcccatggc cagcgaaaca ccaagagttg accccacaga aatctccaac gtcaatgctc
     1321 ctgtgtttag gataatagcg caaatcaaat cacaacccac tgaatctcag ttgattttac
     1381 aatcgccaac catatcatcg aaaaacggca gcgaggttga aatgattacg ttgaacaata
     1441 ttcgtgtatc tatgaataaa acatttgaga tcgactcgtg gtatgagttt gtttgcagga
     1501 acaatgatga cggcgagcta ggatttttga ttctagacgc tgtactatgc aaattcaagg
     1561 agaacgaaga tttaagctta aacggtgtgg ttgctttaca gagactatgt aagaaatacc
     1621 cagaaatata ctagctaaaa tcgcttttat tcttaccttt atgtactgtt gcctctatat
     1681 gtcttccctt catataataa attccatttc taaaaaaatt gtagttgtgt ttatacatgt
     1741 atagatagat aaatggatcc gaattcgagc tcggcgcgcc tgcaggtcga caagcttgcg
     1801 gccgcataat gcttaagtcg aacagaaagt aatcgtattg tacacggccg cataatcgaa
     1861 attaatacga ctcactatag gggaattgtg agcggataac aattccccat cttagtatat
     1921 tagttaagta taagaaggag atatacatat ggcagatctc aattggatat cggccggcca
     1981 cgcgatcgct gacgtcggta ccctcgagtc tggtaaagaa accgctgctg cgaaatttga
     2041 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
     2101 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2161 gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt
     2221 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc
     2281 atcgtggccg gatcttgcgg cccctcggct tgaacgaatt gttagacatt atttgccgac
     2341 taccttggtg atctcgcctt tcacgtagtg gacaaattct tccaactgat ctgcgcgcga
     2401 ggccaagcga tcttcttctt gtccaagata agcctgtcta gcttcaagta tgacgggctg
     2461 atactgggcc ggcaggcgct ccattgccca gtcggcagcg acatccttcg gcgcgatttt
     2521 gccggttact gcgctgtacc aaatgcggga caacgtaagc actacatttc gctcatcgcc
     2581 agcccagtcg ggcggcgagt tccatagcgt taaggtttca tttagcgcct caaatagatc
     2641 ctgttcagga accggatcaa agagttcctc cgccgctgga cctaccaagg caacgctatg
     2701 ttctcttgct tttgtcagca agatagccag atcaatgtcg atcgtggctg gctcgaagat
     2761 acctgcaaga atgtcattgc gctgccattc tccaaattgc agttcgcgct tagctggata
     2821 acgccacgga atgatgtcgt cgtgcacaac aatggtgact tctacagcgc ggagaatctc
     2881 gctctctcca ggggaagccg aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt
     2941 ttcatcaagc cttacggtca ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc
     3001 gccatccact gcggagccgt acaaatgtac ggccagcaac gtcggttcga gatggcgctc
     3061 gatgacgcca actacctctg atagttgagt cgatacttcg gcgatcaccg cttccctcat
     3121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3181 catatttgaa tgtatttaga aaaataaaca aatagctagc tcactcggtc gctacgctcc
     3241 gggcgtgaga ctgcggcggg cgctgcggac acatacaaag ttacccacag attccgtgga
     3301 taagcagggg actaacatgt gaggcaaaac agcagggccg cgccggtggc gtttttccat
     3361 aggctccgcc ctcctgccag agttcacata aacagacgct tttccggtgc atctgtggga
     3421 gccgtgaggc tcaaccatga atctgacagt acgggcgaaa cccgacagga cttaaagatc
     3481 cccaccgttt ccggcgggtc gctccctctt gcgctctcct gttccgaccc tgccgtttac
     3541 cggatacctg ttccgccttt ctcccttacg ggaagtgtgg cgctttctca tagctcacac
     3601 actggtatct cggctcggtg taggtcgttc gctccaagct gggctgtaag caagaactcc
     3661 ccgttcagcc cgactgctgc gccttatccg gtaactgttc acttgagtcc aacccggaaa
     3721 agcacggtaa aacgccactg gcagcagcca ttggtaactg ggagttcgca gaggatttgt
     3781 ttagctaaac acgcggttgc tcttgaagtg tgcgccaaag tccggctaca ctggaaggac
     3841 agatttggtt gctgtgctct gcgaaagcca gttaccacgg ttaagcagtt ccccaactga
     3901 cttaaccttc gatcaaacca cctccccagg tggttttttc gaaggttaag tcagttgggg
     3961 aactgcttaa ccttcgatca aaccacctcc ccaggtggtt ttttcgttta cagggcaaaa
     4021 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta ctgaaccgct
     4081 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
     4141 taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4201 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4261 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4321 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4381 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagt
//
