LOCUS       sequence-464144-        5930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of ubiquitin-activating enzyme 
            E1-like in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5930)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5930)
  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_3ONH
FEATURES             Location/Qualifiers
     source          1..5930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(32..487)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(169..190)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     381..400
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(524..571)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     567..585
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(638..655)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1313..1342)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1365..1387
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1402..1426
                     /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(1426..1445)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1427..1445)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1533..1552)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1758..1835
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1836..2918
                     /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(1855..1874)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3727..3918
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3912..3934)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4020..4159
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4075..4094
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4174..4191)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4345..4933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4425..4444)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5044..5063)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5055..5870
                     /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             5055..5870
                     /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(5128..5147)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
       61 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
      121 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
      181 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
      241 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
      301 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
      361 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
      421 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
      481 ggcgcgtccc attcgccaat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
      541 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
      601 cagcttcctt tcgggctttg ttagcagccg gatctcagtg gtggtggtgg tggtgctcga
      661 gtcattacga acctttaatc agcggcactt ccacgtccgg cagagagcac gtgttgcatg
      721 gcagctcatc atcgacgtcc aggaataatt caatcgcctt gcggatcatg gtatcacctt
      781 cctcgtcgct gaagaggata atggagccat tacccaggtt gatctccgac agggtacgat
      841 cattcaggtc ctcgaagtcg taatcaaaca acagacgctg gttgctagcg tccaacaagc
      901 taatatcttg cggatagctg tatttttcac ggatcagaac aacaaagtcg gacaatttca
      961 tcttgttcag acagtcactt gatagtttta taactcccct acatactcca gagccgccaa
     1021 tctgttcacg gtgggcttca atgatgtcgt tgtcttccat gtccaggtct tccggcgctt
     1081 gatctgcctg gatgcgaata ccgtcataca ggaaacgcag gctatccatt tccttgccct
     1141 gacgtttggc aaacgcttcc atcaggcgac gcagcggcgt ggtctttttg atcttaaaga
     1201 aaatttcaga gctgccgtca ctaactttca ggttaatatg ggtttccggt ttcacttccg
     1261 gcttgacttc cggctttgct tcttggttga cttccgaatc ttgcaggcta ccgtggtgat
     1321 ggtgatggtg atgatggtga tgaatcttcg ccatcatatg tatatctcct tcttaaagtt
     1381 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
     1441 tattaatttc gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg
     1501 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
     1561 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
     1621 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
     1681 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
     1741 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     1801 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     1861 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     1921 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     1981 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     2041 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     2101 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     2161 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     2221 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     2281 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     2341 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     2401 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     2461 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     2521 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     2581 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct
     2641 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     2701 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     2761 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     2821 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     2881 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     2941 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     3001 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     3061 atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc
     3121 tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc
     3181 ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt
     3241 atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     3301 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     3361 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     3421 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     3481 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     3541 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     3601 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     3661 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     3721 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     3781 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     3841 gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat
     3901 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3961 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     4021 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     4081 cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag
     4141 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4201 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4261 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4321 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4381 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4441 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4501 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4561 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4621 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4681 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4741 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4801 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4861 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4921 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4981 aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta
     5041 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa
     5101 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     5161 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     5221 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     5281 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     5341 tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt
     5401 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     5461 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     5521 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     5581 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     5641 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     5701 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     5761 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     5821 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc
     5881 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
//
