LOCUS       sequence-464111-        6518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of isoflavone reductase (IFR) in 
            Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6518)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6518)
  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_2GAS
FEATURES             Location/Qualifiers
     source          1..6518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             99..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(169..187)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      183..230
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      267..722
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(354..373)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     564..585
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(814..1629)
                     /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             complement(814..1629)
                     /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     1537..1556
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1621..1640
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1751..2339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2240..2259
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2493..2510
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2525..2664
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2590..2609)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2750..2772
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2766..2957)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3766..4848)
                     /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     4810..4829
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4849..4926)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5132..5151
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5239..5258
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5239..5257
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5258..5282
                     /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)."
     RBS             5297..5319
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5342..5371
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
ORIGIN
        1 cggcaaagga catcgaggct tctgaggcct atccggacgt gacgtacacc accgcggatg
       61 aatatctgaa tcaatttgtt ggttcgtaat gactcgagca ccaccaccac caccactgag
      121 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
      181 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
      241 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
      301 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      361 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      421 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      481 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      541 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
      601 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
      661 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
      721 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
      781 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
      841 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
      901 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
      961 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     1021 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     1081 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     1141 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     1201 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     1261 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     1321 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     1381 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     1441 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     1501 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     1561 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     1621 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     1681 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     1741 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1801 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1861 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     1921 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1981 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2041 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2101 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2161 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2221 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2281 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2341 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2401 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2461 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2521 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     2581 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     2641 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     2701 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     2761 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     2821 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     2881 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     2941 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3001 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3061 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3121 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     3181 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     3241 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     3301 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     3361 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     3421 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     3481 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     3541 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     3601 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     3661 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     3721 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     3781 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3841 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     3901 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     3961 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4021 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     4081 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     4141 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     4201 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     4261 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     4321 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     4381 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     4441 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     4501 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     4561 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     4621 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     4681 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     4741 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     4801 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     4861 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     4921 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     4981 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     5041 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     5101 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     5161 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     5221 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     5281 cccctctaga aataattttg tttaacttta agaaggagat atacatatga tggcgaagat
     5341 tcatcaccat catcaccatc accatcacca cggtagcctg caagattcgg aagtcaacca
     5401 agaagcaaag ccggaagtca agccggaagt gaaaccggaa acccatatta acctgaaagt
     5461 tagtgacggc agctctgaaa ttttctttaa gatcaaaaag accacgccgc tgcgtcgcct
     5521 gatggaagcg tttgccaaac gtcagggcaa ggaaatggat agcctgcgtt tcctgtatga
     5581 cggtattcgc atccaggcag atcaagcgcc ggaagacctg gacatggaag acaacgacat
     5641 cattgaagcc caccgtgaac agattggcgg ctctggaaca gaaaataaaa tattaattct
     5701 aggacccact ggtgcgattg gccgtcacat tgtttgggca tcgatcaagg cgggtaaccc
     5761 gacctacgca ttggttagaa aaacgattac tgcggcgaat ccggaaacga aagaggagct
     5821 gatcgataat taccagtccc taggcgtgat tttgttggag ggcgacatca acgaccacga
     5881 aaccctggtc aaagcgatca aacaagtcga tatcgtgatc tgcgcggccg gtcgccttct
     5941 gattgaagat caagtgaaga tcatcaaggc gattaaggag gcgggtaacg tcaagaaatt
     6001 cttcccaagc gaatttggtc tggacgttga ccgtcacgat gctgtagagc cggttcgtca
     6061 ggttttcgag gagaaggctt ccatccgccg tgtaattgag gctgaaggtg ttccgtatac
     6121 gtacctgtgt tgccatgcat ttaccggtta tttcctgcgt aacctggcgc aactggacgc
     6181 taccgatccg cctcgtgata aagttgtgat tctgggcgac ggcaacgtta agggcgcgta
     6241 cgttaccgaa gccgacgtgg gtacattcac cattcgcgca gcgaacgacc cgaataccct
     6301 gaacaaagcg gtgcatattc gcttaccgaa aaactacttg acccagaatg aagtgatcgc
     6361 cctgtgggaa aagaaaatcg gcaaaacctt ggagaagacc tacgtgagcg aagagcaggt
     6421 gttgaaagat atccaagaaa gcagctttcc gcacaactac ctcctggctc tgtatcatag
     6481 ccagcagatt aagggtgatg ccgtttatga aatcgacc
//
