LOCUS       sequence-464195-        5921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of RNA-induced transcriptional 
            silencing complex protein tas3 in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5921)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5921)
  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_3D1B
FEATURES             Location/Qualifiers
     source          1..5921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     157..178
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(407..1222)
                     /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(407..1222)
                     /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     1130..1149
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1214..1233
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1344..1932
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1833..1852
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2086..2103
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2118..2257
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2183..2202)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2343..2365
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2359..2550)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3359..4441)
                     /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     4403..4422
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4442..4519)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4725..4744
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4832..4851
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4832..4850
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4851..4875
                     /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             4890..4912
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4935..4964
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5613..5630
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5683..5701)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5697..5744
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5781..5921,1..315)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5868..5887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
       61 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
      121 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
      181 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
      241 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
      301 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
      361 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
      421 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
      481 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
      541 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
      601 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
      661 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
      721 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
      781 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
      841 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
      901 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
      961 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     1021 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     1081 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     1141 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     1201 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     1261 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     1321 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     1381 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     1441 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     1501 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     1561 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     1621 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     1681 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     1741 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     1801 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     1861 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     1921 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     1981 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2041 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2101 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2161 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     2221 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     2281 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     2341 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     2401 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     2461 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     2521 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     2581 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     2641 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     2701 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     2761 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     2821 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     2881 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     2941 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     3001 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     3061 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     3121 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     3181 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     3241 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     3301 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     3361 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3421 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     3481 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     3541 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     3601 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     3661 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     3721 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     3781 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     3841 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     3901 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     3961 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     4021 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     4081 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     4141 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     4201 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     4261 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     4321 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     4381 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     4441 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     4501 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     4561 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     4621 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     4681 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     4741 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     4801 aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     4861 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
     4921 tgatggcgaa gattcatcac catcatcacc atcaccatca ccacggtagc ctgcaagatt
     4981 cggaagtcaa ccaagaagca aagccggaag tcaagccgga agtgaaaccg gaaacccata
     5041 ttaacctgaa agttagtgac ggcagctctg aaattttctt taagatcaaa aagaccacgc
     5101 cgctgcgtcg cctgatggaa gcgtttgcca aacgtcaggg caaggaaatg gatagcctgc
     5161 gtttcctgta tgacggtatt cgcatccagg cagatcaagc gccggaagac ctggacatgg
     5221 aagacaacga catcattgaa gcccaccgtg aacagattgg cggctctgga gatttatata
     5281 taaattggtt gaaatcacta tcctttttcc agacgaacag cagttgcgcg gaagcgctgg
     5341 tgaaagttat tccgcactac cataataagt tgatcgattt cagccaagtt ctgcaactgg
     5401 tctttagcgc ttctgaaaag ttcccgattc aggagaacca gccgctgccg gaacagctga
     5461 tgtttctgtc taatctcgag aagcagaccc catttgccaa agcagttggt agcagcattt
     5521 ataaattggt gaccggcaaa aacctgagcc tggacttcgc gtcgcaaatc cttaaggagg
     5581 cgtccatctt ggagggttcg taatgactcg agcaccacca ccaccaccac tgagatccgg
     5641 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     5701 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     5761 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     5821 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     5881 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt c
//
