LOCUS       sequence-464153-        5981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of RNA binding exosome specificity 
            factor Mmi1 in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5981)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5981)
  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_5EIP
FEATURES             Location/Qualifiers
     source          1..5981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             336..527
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(521..543)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    629..768
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     684..703
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(783..800)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(954..1542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1034..1053)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1653..1672)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1664..2479
                     /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             1664..2479
                     /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(1737..1756)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2571..3026)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2708..2729)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2920..2939
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3063..3110)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3106..3124
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3177..3194)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3903..3932)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             3955..3977
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3992..4016
                     /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(4016..4035)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4017..4035)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4123..4142)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4348..4425
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4426..5508
                     /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(4445..4464)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
       61 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
      121 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
      181 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
      241 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
      301 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
      361 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
      421 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
      481 accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
      541 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
      601 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
      661 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg
      721 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
      781 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      841 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      901 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      961 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1021 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1081 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1141 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1201 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1261 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1321 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1381 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1441 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1501 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1561 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac
     1621 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     1681 ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     1741 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
     1801 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     1861 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     1921 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc
     1981 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     2041 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     2101 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     2161 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     2221 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     2281 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     2341 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     2401 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     2461 gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga
     2521 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     2581 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     2641 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2701 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2761 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2821 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2881 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2941 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     3001 gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc cggatatagt tcctcctttc
     3061 agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca
     3121 gcggtggcag cagccaactc agcttccttt cgggctttgt tagcagccgg atctcagtgg
     3181 tggtggtggt ggtgctcgag tcattacgaa ccaatcaggg tacacagacg agagccttca
     3241 tcatacatca tttccatacc atcacgcgcc tcgctgccat gatccatatg ggacacgatc
     3301 tcggagattt ctgcgctaaa caggttgcac gttttaatcc atttgatacg aacaccaccg
     3361 gcgtggctcg agtcgctcca aaacggcagc tcggtgctat tcaagtcaga aacaacttgc
     3421 gcgtagccca aagcgttata ggtctgctgc gccacgaaga tgaagtacac gctagccttc
     3481 ttgtacgcgt cggagatgcg ctttgagctg tcctttttga ctgcccaaat gctggtcttt
     3541 ttcgcgtggg caatcgcagt ctcgttatca catagcatta taaaatatct tgaccttcca
     3601 gagccgccaa tctgttcacg gtgggcttca atgatgtcgt tgtcttccat gtccaggtct
     3661 tccggcgctt gatctgcctg gatgcgaata ccgtcataca ggaaacgcag gctatccatt
     3721 tccttgccct gacgtttggc aaacgcttcc atcaggcgac gcagcggcgt ggtctttttg
     3781 atcttaaaga aaatttcaga gctgccgtca ctaactttca ggttaatatg ggtttccggt
     3841 ttcacttccg gcttgacttc cggctttgct tcttggttga cttccgaatc ttgcaggcta
     3901 ccgtggtgat ggtgatggtg atgatggtga tgaatcttcg ccatcatatg tatatctcct
     3961 tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta
     4021 tagtgagtcg tattaatttc gcgggatcga gatcgatctc gatcctctac gccggacgca
     4081 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca
     4141 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta
     4201 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc
     4261 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt
     4321 cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt
     4381 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
     4441 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     4501 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     4561 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     4621 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     4681 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     4741 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     4801 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     4861 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     4921 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     4981 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     5041 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     5101 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     5161 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     5221 gcggacatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     5281 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     5341 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     5401 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5461 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     5521 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt
     5581 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
     5641 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg
     5701 cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat
     5761 cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa
     5821 gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt
     5881 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     5941 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg a
//
