LOCUS       sequence-464198-        6113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of replicase polyprotein 1ab (pp1ab)
            (ORF1ab polyprotein) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6113)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6113)
  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_6WEY
FEATURES             Location/Qualifiers
     source          1..6113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(12..827)
                     /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(12..827)
                     /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     735..754
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     819..838
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      949..1537
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1438..1457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1691..1708
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1723..1862
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1788..1807)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1948..1970
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1964..2155)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2964..4046)
                     /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     4008..4027
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4047..4124)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4330..4349
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4437..4456
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4437..4455
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4456..4480
                     /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             4495..4517
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4540..4569
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5410..5427
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5480..5498)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5494..5541
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5578..6033
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5665..5684)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5875..5896
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
       61 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
      121 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
      181 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
      241 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
      301 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
      361 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
      421 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
      481 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
      541 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
      601 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
      661 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
      721 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
      781 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
      841 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
      901 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
      961 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1021 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1081 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1141 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1201 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1261 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     1321 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     1381 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     1441 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     1501 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     1561 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     1621 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     1681 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     1741 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     1801 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     1861 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     1921 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     1981 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     2041 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     2101 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     2161 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     2221 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     2281 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     2341 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     2401 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     2461 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     2521 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     2581 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     2641 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     2701 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     2761 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     2821 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     2881 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     2941 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3001 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     3061 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     3121 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     3181 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     3241 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     3301 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     3361 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     3421 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     3481 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     3541 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     3601 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     3661 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     3721 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     3781 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     3841 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     3901 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     3961 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     4021 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     4081 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     4141 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     4201 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     4261 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     4321 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     4381 gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg cgaaattaat
     4441 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     4501 taactttaag aaggagatat acatatgatg gcgaagattc atcaccatca tcaccatcac
     4561 catcaccacg gtagcctgca agattcggaa gtcaaccaag aagcaaagcc ggaagtcaag
     4621 ccggaagtga aaccggaaac ccatattaac ctgaaagtta gtgacggcag ctctgaaatt
     4681 ttctttaaga tcaaaaagac cacgccgctg cgtcgcctga tggaagcgtt tgccaaacgt
     4741 cagggcaagg aaatggatag cctgcgtttc ctgtatgacg gtattcgcat ccaggcagat
     4801 caagcgccgg aagacctgga catggaagac aacgacatca ttgaagccca ccgtgaacag
     4861 attggcggct ctggaggggt aaattcattt agtggatatc taaaattgac tgacaacgtg
     4921 tacatcaaaa acgcggacat cgtcgaggaa gcaaagaaag ttaaaccgac cgtggtggtg
     4981 aatgcggcaa atgtgtacct caagcacggc ggcggtgttg caggcgcgct gaataaggct
     5041 acgaacaacg ctatgcaggt tgagagcgac gactatatcg ccaccaatgg tccgttgaag
     5101 gtgggcggca gctgcgtgct ctctggtcat aacctggcga aacactgcct gcacgtggtt
     5161 ggtccaaatg tcaacaaggg tgaagatatt cagctgctga aatctgcgta tgagaacttt
     5221 aaccaacatg aggtcttgtt ggctccgtta ctgtccgcgg gtatttttgg tgcggacccg
     5281 attcatagcc tgcgtgtttg tgttgatacc gtacgcacca acgtgtacct ggccgttttc
     5341 gacaagaacc tgtatgacaa actggttagc tcgttcctgg aaatgaaatc cggttcgtaa
     5401 tgactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     5461 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     5521 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     5581 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     5641 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5701 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5761 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5821 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5881 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5941 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     6001 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     6061 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tca
//
