LOCUS       sequence-464180-        6392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of norsolorinic acid synthase (NSAS)
            in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6392)
  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_3ILS
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      11..58
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      95..550
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(182..201)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     392..413
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(642..1457)
                     /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(642..1457)
                     /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     1365..1384
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1449..1468
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1579..2167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2068..2087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2321..2338
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2353..2492
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2418..2437)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2578..2600
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2594..2785)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3594..4676)
                     /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     4638..4657
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4677..4754)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4960..4979
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5067..5086
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5067..5085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5086..5110
                     /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             5125..5147
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5170..5199
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             6319..6336
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(join(6389..6392,1..15))
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
       61 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
      121 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
      181 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
      241 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
      301 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
      361 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
      421 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
      481 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
      541 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      601 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
      661 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
      721 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
      781 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
      841 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
      901 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
      961 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     1021 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     1081 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     1141 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     1201 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1261 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     1321 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     1381 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     1441 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     1501 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1561 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1621 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1681 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     1741 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1801 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1861 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1921 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1981 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2041 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2101 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2161 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     2221 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     2281 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     2341 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2401 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     2461 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     2521 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     2581 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     2641 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     2701 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     2761 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     2821 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     2881 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     2941 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     3001 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     3061 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     3121 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     3181 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     3241 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     3301 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     3361 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     3421 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     3481 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     3541 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     3601 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3661 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     3721 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     3781 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     3841 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     3901 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     3961 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     4021 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     4081 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     4141 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     4201 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     4261 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     4321 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     4381 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     4441 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     4501 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     4561 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     4621 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     4681 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     4741 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     4801 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     4861 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     4921 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     4981 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     5041 cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     5101 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgatg
     5161 gcgaagattc atcaccatca tcaccatcac catcaccacg gtagcctgca agattcggaa
     5221 gtcaaccaag aagcaaagcc ggaagtcaag ccggaagtga aaccggaaac ccatattaac
     5281 ctgaaagtta gtgacggcag ctctgaaatt ttctttaaga tcaaaaagac cacgccgctg
     5341 cgtcgcctga tggaagcgtt tgccaaacgt cagggcaagg aaatggatag cctgcgtttc
     5401 ctgtatgacg gtattcgcat ccaggcagat caagcgccgg aagacctgga catggaagac
     5461 aacgacatca ttgaagccca ccgtgaacag attggcggct ctggactaaa accctattgt
     5521 aggccaagta catcagtcgt gctgcaaggc ctcccgatgg tggcgcgtaa gaccttgttt
     5581 atgctgccgg acggtggcgg ttctgcattc agctacgcgt ccctaccgag attgaaatct
     5641 gacactgcgg ttgtcggcct taattgcccg tacgcgcgcg atccggaaaa catgaactgc
     5701 acccacggcg ctatgattga gagcttctgc aacgagatcc gtcgtcgtca gcctcgtggt
     5761 ccgtaccatc tgggtggctg gtcgagcggt ggtgcgtttg cgtatgtggt cgcagaagca
     5821 ctggttaacc agggcgaaga agttcatagc ctgatcatca ttgatgcccc tattccgcag
     5881 gcaatggaac aactgccgcg tgcattctat gagcactgta atagcatcgg tttgttcgcc
     5941 acccaaccgg gtgcctcgcc ggacggcagc accgaaccgc caagctacct gattccgcat
     6001 tttaccgcgg tggtggatgt tatgctggat tataaactgg cgccactgca cgcccgtcgc
     6061 atgccgaagg tgggtattgt ttgggcggct gacacggtta tggatgagcg cgacgctccg
     6121 aagatgaaag gcatgcattt tatgatccag aaacgtaccg agtttggtcc ggatggttgg
     6181 gataccatta tgccgggcgc ttccttcgac atcgttcgtg ctgacggagc gaatcacttc
     6241 acgctgatgc agaaggagca cgtatccatt atcagcgacc tgatcgaccg cgtgatggcg
     6301 ggttcgtaat gactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     6361 cgaaaggaag ctgagttggc tgctgccacc gc
//
