LOCUS       sequence-464190-        6176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of tripartite motif-containing 
            protein 72 (Mg53) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6176)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6176)
  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_3KB5
FEATURES             Location/Qualifiers
     source          1..6176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(850..879)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             902..924
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    939..963
                     /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(963..982)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(964..982)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1070..1089)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1295..1372
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1373..2455
                     /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(1392..1411)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3264..3455
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3449..3471)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3557..3696
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3612..3631
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3711..3728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3882..4470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3962..3981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4581..4600)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4592..5407
                     /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             4592..5407
                     /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(4665..4684)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5499..5954)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5636..5657)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5848..5867
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5991..6038)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6034..6052
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6105..6122)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 atgccagcac acgtcgaaga acgggtaaac cggacgcggc agacgttcat gaaacgcaaa
       61 taacgggacc aaagcatccg cgtcgctcgc gtcatagaag ctcagcacac cgtcaccgaa
      121 ggacaggtac aggccaatac gcgtcgggcg acgctccggg ctacgcagtg cgcgcggttc
      181 tttcgcctcg acgtgcgctt ccaggatttt accttcacgc aggcccagca gccaaagacc
      241 ttgcgacgga actgcatgca gacgacctct gcgtggcgcc tctgccgcga taacgccgag
      301 tgcccaacgc ggtttgtcgc ccacatcaac ttcccaatag tgctcgccct cggacagttg
      361 ctggtgcgcc accacggcca cagccttgtc aaattggcga gggtcctcac ccgccggtgg
      421 cgctttctgc tcgctacatt ccacgcgacg accgctagag ctaacaacca ggctcgggtg
      481 ggctgaagac gggtcaaagg tcaattcctc caaagcgggc attagtgctc taaacatttt
      541 ccttccagag ccgccaatct gttcacggtg ggcttcaatg atgtcgttgt cttccatgtc
      601 caggtcttcc ggcgcttgat ctgcctggat gcgaataccg tcatacagga aacgcaggct
      661 atccatttcc ttgccctgac gtttggcaaa cgcttccatc aggcgacgca gcggcgtggt
      721 ctttttgatc ttaaagaaaa tttcagagct gccgtcacta actttcaggt taatatgggt
      781 ttccggtttc acttccggct tgacttccgg ctttgcttct tggttgactt ccgaatcttg
      841 caggctaccg tggtgatggt gatggtgatg atggtgatga atcttcgcca tcatatgtat
      901 atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat
      961 tcccctatag tgagtcgtat taatttcgcg ggatcgagat cgatctcgat cctctacgcc
     1021 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
     1081 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
     1141 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
     1201 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
     1261 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
     1321 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     1381 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     1441 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     1501 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     1561 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     1621 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     1681 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     1741 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     1801 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     1861 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     1921 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     1981 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     2041 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     2101 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     2161 cgttggtgcg gacatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     2221 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     2281 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     2341 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2401 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2461 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     2521 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     2581 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     2641 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     2701 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     2761 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
     2821 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     2881 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     2941 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     3001 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     3061 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     3121 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     3181 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     3241 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     3301 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     3361 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     3421 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     3481 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     3541 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     3601 gcttaactat gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata
     3661 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     3721 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3781 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3841 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3901 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3961 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4021 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4081 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4141 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4201 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4261 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4321 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4381 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4441 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4501 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc
     4561 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg
     4621 ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     4681 cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc
     4741 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     4801 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     4861 tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt
     4921 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     4981 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     5041 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     5101 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     5161 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     5221 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     5281 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     5341 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     5401 tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5461 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     5521 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     5581 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     5641 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     5701 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     5761 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     5821 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     5881 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     5941 gctacagggc gcgtcccatt cgccaatccg gatatagttc ctcctttcag caaaaaaccc
     6001 ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc ggtggcagca
     6061 gccaactcag cttcctttcg ggctttgtta gcagccggat ctcagtggtg gtggtggtgg
     6121 tgctcgagtc attacgaacc aaccaacaac agcggctgag cgttcttacc cttatc
//
