LOCUS       sequence-464178-        5930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of 3C-like proteinase in Escherichia
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5930)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5930)
  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: pAIP4_5XBC
FEATURES             Location/Qualifiers
     source          1..5930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     643..662
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     727..746
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      857..1445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1346..1365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1599..1616
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1631..1770
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1696..1715)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1856..1878
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1872..2063)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2872..3954)
                     /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     3916..3935
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3955..4032)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4238..4257
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4345..4364
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4345..4363
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4364..4388
                     /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             4403..4425
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4448..4477
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5135..5152
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5205..5223)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5219..5266
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5303..5758
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5390..5409)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5600..5621
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5850..5930,1..735))
                     /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(join(5850..5930,1..735))
                     /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"
ORIGIN
        1 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
       61 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
      121 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
      181 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
      241 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
      301 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
      361 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
      421 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
      481 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
      541 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
      601 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
      661 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
      721 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
      781 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
      841 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      901 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      961 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1021 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1081 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1141 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1201 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1261 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1321 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1381 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1441 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1501 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1561 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1621 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     1681 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     1741 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     1801 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1861 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     1921 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     1981 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     2041 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     2101 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     2161 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     2221 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     2281 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     2341 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     2401 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     2461 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     2521 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     2581 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     2641 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     2701 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     2761 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     2821 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     2881 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2941 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     3001 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     3061 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     3121 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     3181 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     3241 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     3301 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     3361 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     3421 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     3481 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     3541 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     3601 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     3661 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     3721 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     3781 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     3841 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     3901 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     3961 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     4021 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     4081 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     4141 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     4201 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     4261 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     4321 agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
     4381 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatgatggc
     4441 gaagattcat caccatcatc accatcacca tcaccacggt agcctgcaag attcggaagt
     4501 caaccaagaa gcaaagccgg aagtcaagcc ggaagtgaaa ccggaaaccc atattaacct
     4561 gaaagttagt gacggcagct ctgaaatttt ctttaagatc aaaaagacca cgccgctgcg
     4621 tcgcctgatg gaagcgtttg ccaaacgtca gggcaaggaa atggatagcc tgcgtttcct
     4681 gtatgacggt attcgcatcc aggcagatca agcgccggaa gacctggaca tggaagacaa
     4741 cgacatcatt gaagcccacc gtgaacagat tggcggctct ggagcttcaa atcaagtaac
     4801 actagcgttt gcaaacgacg cggaaattag cgcgtttggc ttctgcaccg catcggaggc
     4861 tgtgtcctat tacagcgaag cggctgcctc cggcttcatg cagtgtcgtt ttgttagctt
     4921 tgacctggca gacacggtag aaggtctgct tccggaagat tacgtgatgg tggtcgttgg
     4981 tactaccaag ctgagcgcgt atgttgatac cttcggctct cgtccgcgca acatctgcgg
     5041 ttggctgctg tttagcaact gcaattactt cctggaggag ttggagttga ccttcggccg
     5101 tcgcggtggt ttagagggtt cgtaatgact cgagcaccac caccaccacc actgagatcc
     5161 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     5221 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     5281 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5341 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5401 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5461 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5521 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5581 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5641 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5701 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     5761 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5821 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     5881 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
//
