LOCUS       sequence-464179-        6161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of cysteine protease avirulence 
            protein AvrPphB in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6161)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6161)
  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_1UKF
FEATURES             Location/Qualifiers
     source          1..6161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(67..86)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     277..298
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(527..1342)
                     /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(527..1342)
                     /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     1250..1269
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1334..1353
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1464..2052
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1953..1972
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2206..2223
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2238..2377
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2303..2322)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2463..2485
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2479..2670)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3479..4561)
                     /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     4523..4542
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4562..4639)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4845..4864
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4952..4971
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4952..4970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4971..4995
                     /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             5010..5032
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5055..5084
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5973..5990
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6043..6061)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6057..6104
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(6141..6161,1..435)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
       61 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
      121 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
      181 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
      241 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
      301 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
      361 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
      421 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
      481 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
      541 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
      601 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
      661 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
      721 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
      781 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
      841 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
      901 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
      961 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     1021 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     1081 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     1141 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     1201 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     1261 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     1321 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     1381 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     1441 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     1501 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     1561 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     1621 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     1681 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     1741 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     1801 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     1861 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     1921 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     1981 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2041 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2101 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2161 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2221 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2281 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     2341 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     2401 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     2461 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     2521 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     2581 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     2641 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     2701 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     2761 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     2821 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     2881 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     2941 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     3001 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     3061 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     3121 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     3181 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     3241 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     3301 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     3361 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     3421 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     3481 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3541 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     3601 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     3661 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     3721 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     3781 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     3841 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     3901 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     3961 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     4021 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     4081 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     4141 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     4201 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     4261 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     4321 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     4381 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     4441 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     4501 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     4561 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     4621 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     4681 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     4741 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     4801 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     4861 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     4921 aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     4981 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
     5041 tgatggcgaa gattcatcac catcatcacc atcaccatca ccacggtagc ctgcaagatt
     5101 cggaagtcaa ccaagaagca aagccggaag tcaagccgga agtgaaaccg gaaacccata
     5161 ttaacctgaa agttagtgac ggcagctctg aaattttctt taagatcaaa aagaccacgc
     5221 cgctgcgtcg cctgatggaa gcgtttgcca aacgtcaggg caaggaaatg gatagcctgc
     5281 gtttcctgta tgacggtatt cgcatccagg cagatcaagc gccggaagac ctggacatgg
     5341 aagacaacga catcattgaa gcccaccgtg aacagattgg cggctctgga tcactatctg
     5401 attttagtgt agctagcagg gacgtgaacc acaacaacat ttgtgcaggc ctgagcacgg
     5461 aatggctggt catgagcagc gacggtgatg cggaatcccg tatggatcac cttgattaca
     5521 acggcgaggg ccaatctcgc ggttctgaac gtcatcaggt ctataacgac gcgctgcgcg
     5581 cagccttgtc taacgacgac gaggctccgt tcttcaccgc gagcactgcg gttattgagg
     5641 acgctggctt cagcctccgc cgtgaaccga agaccgttca cgcgagcggt ggtagcgctc
     5701 aactgggtca aaccgtggcg catgacgtgg cccaatccgg tcgtaagcac ttgctgtcgc
     5761 tgcgttttgc aaatgttcag ggtcatgcga tcgcctgcag ttgcgaaggc tcccagttta
     5821 aattatttga tccgaatctg ggcgagttcc agtccagcag aagcgcggca ccacagctga
     5881 tcaaaggtct gatcgaccac tacaacagct tgaattatga tgtggcgtgc gtgaatgagt
     5941 ttcgtgtttc agttggttcg taatgactcg agcaccacca ccaccaccac tgagatccgg
     6001 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     6061 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     6121 tatccggatt ggcgaatggg acgcgccctg tagcggcgca t
//
