LOCUS       sequence-464120-        6074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of polymerase basic protein 2 
            (RNA-directed RNA polymerase subunit P3) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6074)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6074)
  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_5EF9
FEATURES             Location/Qualifiers
     source          1..6074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     698..717
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     782..801
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      912..1500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1401..1420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1654..1671
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1686..1825
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1751..1770)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1911..1933
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1927..2118)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2927..4009)
                     /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     3971..3990
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4010..4087)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4293..4312
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4400..4419
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4400..4418
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4419..4443
                     /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             4458..4480
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4503..4532
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5334..5351
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5404..5422)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5418..5465
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5502..5957
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5589..5608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5799..5820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6049..6074,1..790))
                     /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(6049..6074,1..790))
                     /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 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
       61 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
      121 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
      181 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
      241 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
      301 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
      361 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
      421 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
      481 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
      541 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
      601 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
      661 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
      721 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
      781 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
      841 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
      901 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
      961 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1021 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     1081 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1141 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1201 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1261 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1321 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1381 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1441 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1501 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1561 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     1621 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     1681 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     1741 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     1801 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     1861 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     1921 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     1981 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     2041 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     2101 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     2161 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     2221 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     2281 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     2341 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     2401 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     2461 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     2521 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     2581 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     2641 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     2701 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     2761 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     2821 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     2881 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     2941 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3001 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3061 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3121 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3181 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     3241 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     3301 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     3361 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     3421 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     3481 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     3541 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     3601 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     3661 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     3721 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     3781 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     3841 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     3901 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     3961 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     4021 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     4081 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     4141 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     4201 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     4261 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     4321 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     4381 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     4441 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg atggcgaaga
     4501 ttcatcacca tcatcaccat caccatcacc acggtagcct gcaagattcg gaagtcaacc
     4561 aagaagcaaa gccggaagtc aagccggaag tgaaaccgga aacccatatt aacctgaaag
     4621 ttagtgacgg cagctctgaa attttcttta agatcaaaaa gaccacgccg ctgcgtcgcc
     4681 tgatggaagc gtttgccaaa cgtcagggca aggaaatgga tagcctgcgt ttcctgtatg
     4741 acggtattcg catccaggca gatcaagcgc cggaagacct ggacatggaa gacaacgaca
     4801 tcattgaagc ccaccgtgaa cagattggcg gctctggaag gcaacgattt ggaagactag
     4861 aattaaaacg cattagcggt cgtggcttca aaaacgacga agagatcttg atcggcaacg
     4921 gcacgattca gaaaatcggc atttgggatg gtgaagagga gttccacgtg cgctgcggtg
     4981 agtgccgtgg catccttaag aagtcccaaa tgcgtatgga aaaactcttg attaacagcg
     5041 caaaaaaaga agatatgaag gacttaatca tcctgtgtat ggtttttagc caagataccc
     5101 gtatgttcca aggtgtgaga ggcgagatta attttctgaa ccgcgcgggt cagctgttgt
     5161 ctccgatgta tcagctgcag cgttacttcc tgaatcgtag caatgacctg tttgaccagt
     5221 ggggttatga agagtccccg aaggcgagcg aactgcatgg tatcaacgaa ctgatgaacg
     5281 cttcggatta caccctgaag ggtgttgtcg tgaccggttc gtaatgactc gagcaccacc
     5341 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     5401 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     5461 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     5521 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5581 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5641 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5701 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5761 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5821 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5881 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5941 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     6001 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     6061 atcgagcatc aaat
//
