LOCUS       sequence-424023-        4499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  deletion of vanillic_acid_shunt.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4499)
  AUTHORS   Ruhl IA, Woodworth SP, Haugen SJ, Alt HM, Beckham GT, Johnson CW
  TITLE     Production of Vanillin From Ferulic Acid by Pseudomonas putida 
            KT2440 Using Metabolic Engineering and In Situ Product Recovery.
  JOURNAL   Microb Biotechnol. 2025 May;18(5):e70152. doi: 
            10.1111/1751-7915.70152.
  PUBMED    40410945
REFERENCE   2  (bases 1 to 4499)
  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"; journalName: "Microb 
            Biotechnol."; date: "2025-05"; volume: "18(5):e70152. doi"; pages: "
            10.1111/1751-7915.70152"
COMMENT     Sequence Label: pIAR015
FEATURES             Location/Qualifiers
     source          1..4499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     601..617
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     601..617
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2147..2164)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2147..2163)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2245..3039
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2299..2318)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2909..2928
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3046..3132,3133..4467)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgcctcaca
      601 caggaaacag ctatgacatg attacgaatt cttcgcgcgc ttcgtcaagg ccacggggta
      661 tgtcacccat gccgagcgcg gtacccgtgt cgaggacgac cctgccctgc ccgacgcgct
      721 gcggataccg ggtgcgatgg tgtttcatca gggtgcggac gtgctcggcc ccggctggca
      781 gttcgtgccc ggcgccaact ggcgacaccc gcaagggccg ggcagcagcc tggccgggct
      841 ggacaaccat ccggtggtgc agatcgccct ggaagatgcc caggcctatg cccgctgggc
      901 aggccgcgaa ctgcccagcg aggcgcagct ggaatacgcc atgcgcggcg gcctgaccga
      961 tgccgacttc agctggggta ccaccgagca gcccaagggc aagctcatgg ccaatacctg
     1021 gcagggtcag ttcccttatc gcaatgcggc gaaggatggt tttaccggta catcgcccgt
     1081 gggttgcttc ccggccaacg gctttggcct gttcgatgcc ggcggcaatg tctgggagct
     1141 gactcgcacg ggctatcggc caggccatga cgcacagcgc gacgccaagc tcgacccctc
     1201 aggcccggcc ctgagtgaca gcttcgaccc ggcagacccc ggcgtgccgg tggcggtaat
     1261 caaaggcggc tcgcacctgt gttcggcgga ccgctgcatg cgctaccgcc cctcggcacg
     1321 ccagccgcag ccggtgttca tgacgacctc gcacgtgggt ttcagaacga ttcggcaatg
     1381 agatatcctg gacgatggcc gtgtcctcga agacggtgca cagcccggcc gcactcaagg
     1441 ccgcaccttg gcgtgcaatg cgtcggggtc ttcgccacgg tacagcgcct cgacctgagc
     1501 ggcgcgccgc tgcagcacgg cgcgctgatt gatcgagccc ttgtcggtga tttcaccggc
     1561 gtcgatcgac ggcggctcgg ccagcagcga cagccattcg atacggctgg cgttgccttg
     1621 ggcatcgcgg ttcaagcgct ccagccagtc agcgaaccaa ctgcgcacgg tgtcgttggc
     1681 cagcacccgc gcatcgctgg catcctctgc cagcccggcc aggcgccgac actcgggcag
     1741 acgcgggaac accagcaggc ccaggcattc acggtccggc gcggtgacca cgatgtcctg
     1801 tacgtaaggc gagccctcca gcactgcgcg gttgcgcagc ggcccgacac tgacgaatac
     1861 cccggacgaa agtttgaagt cctcagcgat acggccatcg aacatcaggc caagctcggg
     1921 ctgcctggca tcggccagct tcaacgcgtc gcccgaacag tagaagccct cctcgtcgaa
     1981 cgcctcggcg gtctgctgcg gcgagcgcca gtagcccggc atgatatgcg ggccacggaa
     2041 gcgcgcctcg agcttgtcgc ccaccggcac cagcttcact tcgcagccag gtgccggcag
     2101 cccgacatag ccggccatcg acaaaggccc ggtggtgaag cttggcactg gccgtcgttt
     2161 tacaacgtcg tgaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg
     2221 caaacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     2281 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     2341 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     2401 ctgtccggtg ccctgaatga actccaagac gaggcagcgc ggctatcgtg gctggccacg
     2461 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     2521 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     2581 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     2641 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     2701 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     2761 aggctcaagg cgcggatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     2821 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     2881 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     2941 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     3001 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgacgatgaa catcaaaaag
     3061 tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact
     3121 caagcgtttg cgaaagaaac gaaccaaaag ccatataagg aaacatacgg catttcccat
     3181 attacacgcc atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa atatcaagtt
     3241 tctgaatttg attcgtccac aattaaaaat atctcttctg caaaaggcct ggacgtttgg
     3301 gacagctggc cattacaaaa cgctgacggc actgtcgcaa actatcacgg ctaccacatc
     3361 gtctttgcat tagccggaga tcctaaaaat gcggatgaca catcgattta catgttctat
     3421 caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt ctttaaagac
     3481 agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga atggtcaggt
     3541 tcagccacat ttacatctga cggaaaaatc cgtttattct acactgattt ctccggtaaa
     3601 cattacggca aacaaacact gacaactgca caagttaacg tatcagcatc agacagctct
     3661 ttgaacatca acggtgtaga ggattataaa tcaatctttg acggtgacgg aaaaacgtat
     3721 caaaatgtac agcagttcat cgatgaaggc aactacagct caggcgacaa ccatacgctg
     3781 agagatcctc actacgtaga agataaaggc cacaaatact tagtatttga agcaaacact
     3841 ggaactgaag atggctacca aggcgaagaa tctttattta acaaagcata ctatggcaaa
     3901 agcacatcat tcttccgtca agaaagtcaa aaacttctgc aaagcgataa aaaacgcacg
     3961 gctgagttag caaacggcgc tctcggtatg attgagctaa acgatgatta cacactgaaa
     4021 aaagtgatga aaccgctgat tgcatctaac acagtaacag atgaaattga acgcgcgaac
     4081 gtctttaaaa tgaacggcaa atggtacctg ttcactgact cccgcggatc aaaaatgacg
     4141 attgacggca ttacgtctaa cgatatttac atgcttggtt atgtttctaa ttctttaact
     4201 ggcccataca agccgctgaa caaaactggc cttgtgttaa aaatggatct tgatcctaac
     4261 gatgtaacct ttacttactc acacttcgct gtacctcaag cgaaaggaaa caatgtcgtg
     4321 attacaagct atatgacaaa cagaggattc tacgcagaca aacaatcaac gtttgcgccg
     4381 agcttcctgc tgaacatcaa aggcaagaaa acatctgttg tcaaagacag catccttgaa
     4441 caaggacaat taacagttaa caaataatca gaccccgtag aaaagatcaa aggatcttc
//
