LOCUS       sequence-464167-        5960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of putative cupin_2 
            domain-containing isomerase in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5960)
  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_5NJO
FEATURES             Location/Qualifiers
     source          1..5960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(637..666)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             689..711
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    726..750
                     /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(750..769)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(751..769)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(857..876)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1082..1159
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1160..2242
                     /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(1179..1198)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3051..3242
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3236..3258)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3344..3483
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3399..3418
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3498..3515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3669..4257)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3749..3768)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4368..4387)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4379..5194
                     /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             4379..5194
                     /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(4452..4471)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5286..5741)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5423..5444)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5635..5654
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5778..5825)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5821..5839
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5892..5909)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gtgacgttca tcctgttcgt taaacgccgg agccaccgtc agaaccattt ccatgctgcc
       61 gaagtaatga atacgggtat ttggcggcac ggtgatcaag tcggtggcac ccgccgggac
      121 cgcctcacta ttcaggtaaa aaacaccttg accgctaaca acgtaatagg tatagctgct
      181 tctcacgttg aagaactctt gaaaatgacc acgttcaaca gacacgcgta ccacggttgc
      241 gctcggatca acctggccgt agacagtcag gtcaatgccg tgtttgttga agcgctcgga
      301 ttcctctaca gtatacttgt attttgtcat tccagagccg ccaatctgtt cacggtgggc
      361 ttcaatgatg tcgttgtctt ccatgtccag gtcttccggc gcttgatctg cctggatgcg
      421 aataccgtca tacaggaaac gcaggctatc catttccttg ccctgacgtt tggcaaacgc
      481 ttccatcagg cgacgcagcg gcgtggtctt tttgatctta aagaaaattt cagagctgcc
      541 gtcactaact ttcaggttaa tatgggtttc cggtttcact tccggcttga cttccggctt
      601 tgcttcttgg ttgacttccg aatcttgcag gctaccgtgg tgatggtgat ggtgatgatg
      661 gtgatgaatc ttcgccatca tatgtatatc tccttcttaa agttaaacaa aattatttct
      721 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
      781 tcgagatcga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
      841 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
      901 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
      961 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
     1021 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     1081 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     1141 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     1201 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     1261 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     1321 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     1381 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1441 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1501 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1561 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1621 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     1681 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     1741 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     1801 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     1861 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     1921 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga
     1981 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     2041 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     2101 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     2161 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2221 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     2281 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2341 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     2401 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     2461 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     2521 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     2581 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     2641 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     2701 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     2761 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     2821 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     2881 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     2941 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     3001 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     3061 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     3121 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     3181 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3241 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3301 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3361 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     3421 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3481 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3541 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3601 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3661 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3721 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3781 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3841 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3901 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3961 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4021 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4081 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4141 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4201 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4261 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4321 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     4381 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     4441 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     4501 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     4561 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     4621 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     4681 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     4741 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     4801 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     4861 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     4921 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     4981 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5041 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5101 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5161 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     5221 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5281 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5341 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5401 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5461 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5521 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5581 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5641 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     5701 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc caatccggat
     5761 atagttcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat
     5821 gctagttatt gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca
     5881 gccggatctc agtggtggtg gtggtggtgc tcgagtcatt acgaaccata cggggactcc
     5941 gactcgctga taaaacgcac
//
