LOCUS       sequence-464170-        6092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of replicase polyprotein 1a (pp1a) 
            (ORF1a polyprotein) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6092)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6092)
  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_5HIH
FEATURES             Location/Qualifiers
     source          1..6092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..43)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     234..253
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(377..424)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     420..438
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(491..508)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1328..1357)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1380..1402
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1417..1441
                     /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     complement(1441..1460)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1442..1460)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1548..1567)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1773..1850
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1851..2933
                     /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(1870..1889)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3742..3933
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3927..3949)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4035..4174
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4090..4109
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4189..4206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4360..4948)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4440..4459)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5059..5078)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5070..5885
                     /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             5070..5885
                     /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"
     primer_bind     complement(5143..5162)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5977..6092,1..340))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
       61 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
      121 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
      181 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
      241 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
      301 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc aatccggata
      361 tagttcctcc tttcagcaaa aaacccctca agacccgttt agaggcccca aggggttatg
      421 ctagttattg ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag
      481 ccggatctca gtggtggtgg tggtggtgct cgagtcatta cgaaccgatg gtcagggatt
      541 tatacacatc ctgggagttg acgacaacca aaacgcgagt cttcgcctca cgaatcaggt
      601 aatcgaacga cacagccggt ttaacgccaa agatgcctgc gctgaccaat ggggtcacaa
      661 ccaacggata cgcattcata gctttgtagc atttgctcag cagagaaacg tcctgcttcg
      721 cacgcgcatc cggacccacc acgtgcagaa tgttcttggc gaggctgtga ccttgcagta
      781 acacgctgtc gcccacttgc agcggaccct tcgccaggat gtattcgtcg ctctcttttt
      841 gcaccgcgcc cttgctagct gcgttgatgg cacccgcaat gccaccacca tgtttcagat
      901 gggtatttgc cgcgttcacc aaaacagatt caccatagca ctttgcaacc tggatcgcgt
      961 cgcccagaac aatcgtaaca cactcggtga ttactttgtg ttcaaaattt gataggggat
     1021 ctccagagcc gccaatctgt tcacggtggg cttcaatgat gtcgttgtct tccatgtcca
     1081 ggtcttccgg cgcttgatct gcctggatgc gaataccgtc atacaggaaa cgcaggctat
     1141 ccatttcctt gccctgacgt ttggcaaacg cttccatcag gcgacgcagc ggcgtggtct
     1201 ttttgatctt aaagaaaatt tcagagctgc cgtcactaac tttcaggtta atatgggttt
     1261 ccggtttcac ttccggcttg acttccggct ttgcttcttg gttgacttcc gaatcttgca
     1321 ggctaccgtg gtgatggtga tggtgatgat ggtgatgaat cttcgccatc atatgtatat
     1381 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     1441 ccctatagtg agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg
     1501 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     1561 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     1621 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     1681 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     1741 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     1801 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     1861 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     1921 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     1981 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     2041 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     2101 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     2161 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     2221 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     2281 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     2341 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     2401 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     2461 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     2521 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
     2581 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
     2641 ttggtgcgga catctcggta gtgggatacg acgataccga agacagctca tgttatatcc
     2701 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
     2761 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
     2821 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2881 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     2941 gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga
     3001 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     3061 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     3121 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     3181 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     3241 gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg
     3301 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     3361 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     3421 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     3481 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     3541 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     3601 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     3661 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     3721 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     3781 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     3841 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc
     3901 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     3961 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     4021 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
     4081 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc
     4141 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     4201 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4261 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4321 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4381 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4441 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4501 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4561 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4621 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4681 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4741 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4801 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4861 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4921 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4981 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg
     5041 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct
     5101 ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     5161 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag
     5221 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     5281 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     5341 ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc caggtattag
     5401 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     5461 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     5521 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     5581 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     5641 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     5701 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     5761 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     5821 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     5881 tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5941 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     6001 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     6061 gcaaaatccc ttataaatca aaagaataga cc
//
