LOCUS       sequence-464177-        6098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of Wufb25h12 in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6098)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6098)
  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_4ACJ
FEATURES             Location/Qualifiers
     source          1..6098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             266..457
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(451..473)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    559..698
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     614..633
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(713..730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(884..1472)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(964..983)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1583..1602)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1594..2409
                     /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             1594..2409
                     /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(1667..1686)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2501..2956)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2638..2659)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2850..2869
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2993..3040)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3036..3054
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3107..3124)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3950..3979)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             4002..4024
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4039..4063
                     /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(4063..4082)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4064..4082)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4170..4189)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4395..4472
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4473..5555
                     /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(4492..4511)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
       61 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
      121 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
      181 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
      241 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
      301 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
      361 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
      421 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
      481 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
      541 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
      601 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa
      661 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
      721 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
      781 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
      841 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      901 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
      961 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1021 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1081 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1141 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1201 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1261 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1321 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     1381 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1441 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     1501 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg
     1561 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     1621 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1681 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     1741 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1801 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1861 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     1921 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     1981 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     2041 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     2101 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     2161 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     2221 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2281 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     2341 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     2401 tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2461 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     2521 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2581 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2641 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2701 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2761 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2821 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2881 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2941 ccgctacagg gcgcgtccca ttcgccaatc cggatatagt tcctcctttc agcaaaaaac
     3001 ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag
     3061 cagccaactc agcttccttt cgggctttgt tagcagccgg atctcagtgg tggtggtggt
     3121 ggtgctcgag tcattacgaa ccctcaaaag accagatctc aatatcctgg acaaagaaat
     3181 cttctttcat gctcaacatc ggattgccga acgtcttgca gctatggtta cgaccatggt
     3241 acaggtcgcc gtctaaccac aggccgaact caccggaacc accgccgaag gccagagaat
     3301 ccatgtcacc cttgatgaaa aacaggttgt cgccggtcca tttatacgcc tcgaattccg
     3361 ggtagaaggt gaacaggaag gtttcacccg taccataaaa accttcgcta actttaaacg
     3421 gttcgctcgc cagtgcgcca aagatttggc catcggaatc cttaatcacc agcaacatcg
     3481 gcgagtcctg gtcctgcatt gcgcggtaga gagtcttaat gctcatgccg tgcttggagg
     3541 tgctaaacgc caagttccac ggataaccaa tggtacgtgg cggcaggtgt ttagccagtt
     3601 tctcgatttg ctcagcctct aatagatctg agggttcatt tcctccagag ccgccaatct
     3661 gttcacggtg ggcttcaatg atgtcgttgt cttccatgtc caggtcttcc ggcgcttgat
     3721 ctgcctggat gcgaataccg tcatacagga aacgcaggct atccatttcc ttgccctgac
     3781 gtttggcaaa cgcttccatc aggcgacgca gcggcgtggt ctttttgatc ttaaagaaaa
     3841 tttcagagct gccgtcacta actttcaggt taatatgggt ttccggtttc acttccggct
     3901 tgacttccgg ctttgcttct tggttgactt ccgaatcttg caggctaccg tggtgatggt
     3961 gatggtgatg atggtgatga atcttcgcca tcatatgtat atctccttct taaagttaaa
     4021 caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat
     4081 taatttcgcg ggatcgagat cgatctcgat cctctacgcc ggacgcatcg tggccggcat
     4141 caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga
     4201 tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc
     4261 cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt
     4321 gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga
     4381 gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc
     4441 gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg
     4501 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg
     4561 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca
     4621 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca
     4681 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac
     4741 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg
     4801 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg
     4861 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg
     4921 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg
     4981 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa
     5041 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa
     5101 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca
     5161 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg
     5221 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gacatctcgg
     5281 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca
     5341 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg
     5401 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc
     5461 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     5521 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag
     5581 ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc
     5641 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc
     5701 atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt
     5761 cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc
     5821 gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc
     5881 gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     5941 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     6001 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     6061 cgtgtttcgt aaagtctgga aacgcggaag tcagcgcc
//
