LOCUS       sequence-431365-        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"
     CDS             complement(843..1637)
                     /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(954..973)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1564..1583
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1718..1735
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1719..1735
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3265..3281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3265..3281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(3293..3881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3373..3392)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3914..4499,1..749,750..836))
                     /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 ttaaataaag attcttcgcc ttggtagcca tcttcagttc cagtgtttgc ttcaaatact
       61 aagtatttgt ggcctttatc ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct
      121 gagctgtagt tgccttcatc gatgaactgc tgtacatttt gatacgtttt tccgtcaccg
      181 tcaaagattg atttataatc ctctacaccg ttgatgttca aagagctgtc tgatgctgat
      241 acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg
      301 tagaataaac ggatttttcc gtcagatgta aatgtggctg aacctgacca ttcttgtgtt
      361 tggtctttta ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca
      421 gcgtttttcc agctgtcaat agaagtttcg ccgacttttt gatagaacat gtaaatcgat
      481 gtgtcatccg catttttagg atctccggct aatgcaaaga cgatgtggta gccgtgatag
      541 tttgcgacag tgccgtcagc gttttgtaat ggccagctgt cccaaacgtc caggcctttt
      601 gcagaagaga tatttttaat tgtggacgaa tcaaattcag aaacttgata tttttcattt
      661 ttttgctgtt cagggatttg cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt
      721 tccttatatg gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc
      781 agtgcggtag taaaggttaa tactgttgct tgttttgcaa actttttgat gttcatcgtc
      841 gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
      901 ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
      961 cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
     1021 aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
     1081 acgacgagat cctcgccgtc gggcatccgc gccttgagcc tggcgaacag ttcggctggc
     1141 gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga
     1201 gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca
     1261 agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
     1321 tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
     1381 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
     1441 cgcgctgcct cgtcttggag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
     1501 accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
     1561 tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
     1621 ccatcttgtt caatcatgcg aaacgatcct catcctgttt gcagggcttc ccaaccttac
     1681 cagagggcgc cccagctggc aattccggtc acgacgttgt aaaacgacgg ccagtgccaa
     1741 gcttcaccac cgggcctttg tcgatggccg gctatgtcgg gctgccggca cctggctgcg
     1801 aagtgaagct ggtgccggtg ggcgacaagc tcgaggcgcg cttccgtggc ccgcatatca
     1861 tgccgggcta ctggcgctcg ccgcagcaga ccgccgaggc gttcgacgag gagggcttct
     1921 actgttcggg cgacgcgttg aagctggccg atgccaggca gcccgagctt ggcctgatgt
     1981 tcgatggccg tatcgctgag gacttcaaac tttcgtccgg ggtattcgtc agtgtcgggc
     2041 cgctgcgcaa ccgcgcagtg ctggagggct cgccttacgt acaggacatc gtggtcaccg
     2101 cgccggaccg tgaatgcctg ggcctgctgg tgttcccgcg tctgcccgag tgtcggcgcc
     2161 tggccgggct ggcagaggat gccagcgatg cgcgggtgct ggccaacgac accgtgcgca
     2221 gttggttcgc tgactggctg gagcgcttga accgcgatgc ccaaggcaac gccagccgta
     2281 tcgaatggct gtcgctgctg gccgagccgc cgtcgatcga cgccggtgaa atcaccgaca
     2341 agggctcgat caatcagcgc gccgtgctgc agcggcgcgc cgctcaggtc gaggcgctgt
     2401 accgtggcga agaccccgac gcattgcacg ccaaggtgcg gccttgagtg cggccgggct
     2461 gtgcaccgtc ttcgaggaca cggccatcgt ccaggatatc tcattgccga atcgttctga
     2521 aacccacgtg cgaggtcgtc atgaacaccg gctgcggctg gcgtgccgag gggcggtagc
     2581 gcatgcagcg gtccgccgaa cacaggtgcg agccgccttt gattaccgcc accggcacgc
     2641 cggggtctgc cgggtcgaag ctgtcactca gggccgggcc tgaggggtcg agcttggcgt
     2701 cgcgctgtgc gtcatggcct ggccgatagc ccgtgcgagt cagctcccag acattgccgc
     2761 cggcatcgaa caggccaaag ccgttggccg ggaagcaacc cacgggcgat gtaccggtaa
     2821 aaccatcctt cgccgcattg cgataaggga actgaccctg ccaggtattg gccatgagct
     2881 tgcccttggg ctgctcggtg gtaccccagc tgaagtcggc atcggtcagg ccgccgcgca
     2941 tggcgtattc cagctgcgcc tcgctgggca gttcgcggcc tgcccagcgg gcataggcct
     3001 gggcatcttc cagggcgatc tgcaccaccg gatggttgtc cagcccggcc aggctgctgc
     3061 ccggcccttg cgggtgtcgc cagttggcgc cgggcacgaa ctgccagccg gggccgagca
     3121 cgtccgcacc ctgatgaaac accatcgcac ccggtatccg cagcgcgtcg ggcagggcag
     3181 ggtcgtcctc gacacgggta ccgcgctcgg catgggtgac ataccccgtg gccttgacga
     3241 agcgcgcgaa gaattcgtaa tcatgtcata gctgtttcct gtgtgaggcg tttttccata
     3301 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3361 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3421 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3481 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3541 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3601 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3661 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3721 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3781 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3841 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3901 ctacggggtc tgattatttg ttaactgtta attgtccttg ttcaaggatg ctgtctttga
     3961 caacagatgt tttcttgcct ttgatgttca gcaggaagct cggcgcaaac gttgattgtt
     4021 tgtctgcgta gaatcctctg tttgtcatat agcttgtaat cacgacattg tttcctttcg
     4081 cttgaggtac agcgaagtgt gagtaagtaa aggttacatc gttaggatca agatccattt
     4141 ttaacacaag gccagttttg ttcagcggct tgtatgggcc agttaaagaa ttagaaacat
     4201 aaccaagcat gtaaatatcg ttagacgtaa tgccgtcaat cgtcattttt gatccgcggg
     4261 agtcagtgaa caggtaccat ttgccgttca ttttaaagac gttcgcgcgt tcaatttcat
     4321 ctgttactgt gttagatgca atcagcggtt tcatcacttt tttcagtgtg taatcatcgt
     4381 ttagctcaat cataccgaga gcgccgtttg ctaactcagc cgtgcgtttt ttatcgcttt
     4441 gcagaagttt ttgactttct tgacggaaga atgatgtgct tttgccatag tatgctttg
//
