LOCUS       sequence-464196-        6044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of ubiquitin-conjugating enzyme E2 
            D2 in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6044)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6044)
  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_2ESK
FEATURES             Location/Qualifiers
     source          1..6044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             411..602
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(596..618)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    704..843
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     759..778
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(858..875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1029..1617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1109..1128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1728..1747)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1739..2554
                     /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             1739..2554
                     /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(1812..1831)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2646..3101)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2783..2804)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2995..3014
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3138..3185)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3181..3199
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3252..3269)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4041..4070)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             4093..4115
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4130..4154
                     /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(4154..4173)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4155..4173)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4261..4280)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4486..4563
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4564..5646
                     /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(4583..4602)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
       61 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
      121 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
      181 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
      241 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
      301 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
      361 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
      421 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
      481 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
      541 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
      601 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
      661 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
      721 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
      781 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
      841 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
      901 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      961 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1021 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1081 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1141 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1201 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1261 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1321 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     1381 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     1441 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     1501 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     1561 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     1621 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     1681 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     1741 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     1801 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     1861 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     1921 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     1981 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     2041 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     2101 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     2161 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     2221 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     2281 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     2341 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     2401 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     2461 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     2521 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     2581 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     2641 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2701 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2761 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2821 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     2881 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     2941 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3001 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     3061 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc caatccggat
     3121 atagttcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat
     3181 gctagttatt gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca
     3241 gccggatctc agtggtggtg gtggtggtgc tcgagtcatt acgaacccat agcgtatttt
     3301 tgggtccact cgcgagcaat acgattgtac ttttcacgat cggttttata gatgcgcgca
     3361 atttccggaa ccaacgggtc gtccgggttc ggatcgcaaa gcaggctgca gatggacaat
     3421 aacaccttgc tgatcgtcag cgccgggctc cattgcgaac gcagaatatc caggcaaatg
     3481 ctgccattgg agttaatgtt cgggtggtag atacgggtgg tgaaggccac ctttggcggt
     3541 ttgaacggat agtcagtcgg aaagtggatc gtcaggaaaa agacaccacc ctgatacggg
     3601 ctgtcgttcg gacccatgat ggtcgcctgc caatgaaaca tgtcgtcacc aactgggcct
     3661 gcgctacact gcgcaggcgg atctcttgcc aggtcattca gctctttgtg tatcctcttt
     3721 agtgccatag ctcctccaga gccgccaatc tgttcacggt gggcttcaat gatgtcgttg
     3781 tcttccatgt ccaggtcttc cggcgcttga tctgcctgga tgcgaatacc gtcatacagg
     3841 aaacgcaggc tatccatttc cttgccctga cgtttggcaa acgcttccat caggcgacgc
     3901 agcggcgtgg tctttttgat cttaaagaaa atttcagagc tgccgtcact aactttcagg
     3961 ttaatatggg tttccggttt cacttccggc ttgacttccg gctttgcttc ttggttgact
     4021 tccgaatctt gcaggctacc gtggtgatgg tgatggtgat gatggtgatg aatcttcgcc
     4081 atcatatgta tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat
     4141 ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tcgatctcga
     4201 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     4261 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     4321 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     4381 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     4441 gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg
     4501 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     4561 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     4621 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     4681 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     4741 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     4801 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     4861 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     4921 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     4981 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     5041 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     5101 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     5161 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     5221 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     5281 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     5341 tccgggctgc gcgttggtgc ggacatctcg gtagtgggat acgacgatac cgaagacagc
     5401 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     5461 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     5521 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     5581 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     5641 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc
     5701 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     5761 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     5821 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     5881 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     5941 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat
     6001 gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggtt
//
