LOCUS       sequence-464115-        6605 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of o-succinylbenzoate synthase (OSB 
            synthase) in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6605)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6605)
  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_2OKT
FEATURES             Location/Qualifiers
     source          1..6605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             143..160
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(213..231)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      227..274
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      311..766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(398..417)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     608..629
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(858..1673)
                     /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(858..1673)
                     /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     1581..1600
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1665..1684
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1795..2383
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2284..2303
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2537..2554
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2569..2708
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2634..2653)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2794..2816
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2810..3001)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3810..4892)
                     /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     4854..4873
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4893..4970)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5176..5195
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5283..5302
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5283..5301
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5302..5326
                     /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             5341..5363
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5386..5415
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
ORIGIN
        1 tgttacgccg gcgggttatt acttcgagca agatgttgtg gctcacagcg gtatcctgaa
       61 agaaggccgt ctggagttca gacctccgct ggttgacatc acccaattac aaccgtatga
      121 gggcggttcg taatgactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
      181 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
      241 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
      301 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
      361 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
      421 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      481 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
      541 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
      601 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
      661 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
      721 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
      781 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
      841 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
      901 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
      961 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     1021 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     1081 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     1141 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     1201 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     1261 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     1321 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     1381 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     1441 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     1501 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     1561 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     1621 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     1681 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     1741 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1801 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1861 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1921 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     1981 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2041 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2101 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2161 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2221 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2281 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2341 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2401 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2461 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2521 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     2581 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     2641 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     2701 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2761 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     2821 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     2881 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     2941 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     3001 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     3061 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     3121 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     3181 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     3241 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     3301 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     3361 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     3421 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     3481 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     3541 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     3601 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     3661 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     3721 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     3781 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3841 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3901 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     3961 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4021 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4081 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     4141 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     4201 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     4261 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     4321 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     4381 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     4441 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     4501 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     4561 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     4621 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     4681 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     4741 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     4801 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     4861 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     4921 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     4981 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     5041 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     5101 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     5161 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     5221 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa
     5281 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     5341 tttgtttaac tttaagaagg agatatacat atgatggcga agattcatca ccatcatcac
     5401 catcaccatc accacggtag cctgcaagat tcggaagtca accaagaagc aaagccggaa
     5461 gtcaagccgg aagtgaaacc ggaaacccat attaacctga aagttagtga cggcagctct
     5521 gaaattttct ttaagatcaa aaagaccacg ccgctgcgtc gcctgatgga agcgtttgcc
     5581 aaacgtcagg gcaaggaaat ggatagcctg cgtttcctgt atgacggtat tcgcatccag
     5641 gcagatcaag cgccggaaga cctggacatg gaagacaacg acatcattga agcccaccgt
     5701 gaacagattg gcggctctgg atcattaaaa ttgacagctc tacactttta taagtattcg
     5761 gaaccgttca aatctcagat cgtgaccccg aaggtcaccc tgacccatcg tgattgtctg
     5821 tttatcgaac tgatcgacga caaaggaaac gcgtactttg gtgaatgcaa cgcgtttcag
     5881 accgattggt atgatcacga aaccattgcc tccgtgaagc acgtgatcga gcaatggttt
     5941 gaagataacc gcaacaaatc gttcgagacg tacgaggcgg ctctcaagct ggtcgacagc
     6001 ctggagaaca ccccggcagc gcgtgcaacc atcgtgatgg cactgtacca gatgtttcac
     6061 gttctgccga gctttagcgt tgcctacggc gcaactgcgt ccggtctgag caataagcag
     6121 cttgagagct tgaaggcgac caaaccgacg cgtattaaac tcaagtggac cccacagatt
     6181 atgcatcaga ttcgtgtact gcgtgagttg gacttccact ttcaattggt tattgacgcc
     6241 aatgaaagct tggaccgtca ggatttcacc caattgcaat tgctagctcg cgaacaggtt
     6301 ttgtacattg aggaaccgtt taaagatatt agcatgctgg acgaagttgc ggatggcacc
     6361 atcccgccta ttgcgctgga tgaaaaagca acttcactgc tggacatcat caacctgatt
     6421 gagctgtaca atgttaaggt ggtggttctc aagccgttcc gcctgggtgg tatcgacaaa
     6481 gtccagactg cgattgatac cctgaagtcc catggcgcga aagtggtgat cggcggcatg
     6541 tatgagtatg gtctgtctcg ttatttcacg gctatgttag cgcgcaaagg tgactacccg
     6601 ggtga
//
