LOCUS       sequence-431354-        4494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  deletion of areA(calA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4494)
  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 4494)
  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: pIAR004
FEATURES             Location/Qualifiers
     source          1..4494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      698..1286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1187..1206
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1298..1314
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1298..1314
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2839..2856)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2839..2855)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2937..3731
                     /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(2991..3010)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3601..3620
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3738..3824,3825..4494,1..665)
                     /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 ataaaggcca caaatactta gtatttgaag caaacactgg aactgaagat ggctaccaag
       61 gcgaagaatc tttatttaac aaagcatact atggcaaaag cacatcattc ttccgtcaag
      121 aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc tgagttagca aacggcgctc
      181 tcggtatgat tgagctaaac gatgattaca cactgaaaaa agtgatgaaa ccgctgattg
      241 catctaacac agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg aacggcaaat
      301 ggtacctgtt cactgactcc cgcggatcaa aaatgacgat tgacggcatt acgtctaacg
      361 atatttacat gcttggttat gtttctaatt ctttaactgg cccatacaag ccgctgaaca
      421 aaactggcct tgtgttaaaa atggatcttg atcctaacga tgtaaccttt acttactcac
      481 acttcgctgt acctcaagcg aaaggaaaca atgtcgtgat tacaagctat atgacaaaca
      541 gaggattcta cgcagacaaa caatcaacgt ttgcgccgag cttcctgctg aacatcaaag
      601 gcaagaaaac atctgttgtc aaagacagca tccttgaaca aggacaatta acagttaaca
      661 aataatcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
      721 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
      781 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
      841 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
      901 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      961 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1021 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1081 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1141 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1201 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     1261 cgtcaggggg gcggagccta tggaaaaacg cctcacacag gaaacagcta tgacatgatt
     1321 acgaattccc ctgttgctcc accagtgccc gcaacacacc ggcctgcggg ccgcgcagcg
     1381 cggtatacag cacctcgcgt acgcgcgccg ggcccagtac cttggcgtca agcgggtcct
     1441 gcaggcagtg cagcagccgt tccacacttt cgcgcatggg tgcatccagt gccgccgagg
     1501 tcatcgattg cggggtttgc gcctgcactt cagcgtccgg tgccaagccc atgctctgca
     1561 ccaactcgcc cagcacgcca cggtcgatgc ccaccgccac gcccaacagc ggcgcctcgc
     1621 gggtggcaaa ggtttcacac atgaacggca ccgacaacgc ctgcaccagg taatgccctg
     1681 caccgtactc cagggtacgc ggccccaggc gtgccagctt gctgccctgg gccacgatca
     1741 tgaggctggg ttcgtaaatc tgtgggctgc tggccacata gtggtcccag ctcaacacct
     1801 gcacttgggg caggagggtg ggcacgaagc ccgggcgcgt ggtgaggctg cggatcagtg
     1861 agcagagcgg cgcattggca tcgacgtgac gggtcagttg catggtggac caagggcaga
     1921 gaatcagaca gcttgatcat cgcagattga gggcatggcg ccagccatgg cttggcatct
     1981 tcgtagaatc aggcatgcat gccggagaat cagtcgtggg cgaggcggcg aacgacgcgc
     2041 aaaatgtgtc gcctgaatcg tttcacttca ctgcgaggga tatcttgatc ggttgcctgt
     2101 acgggcctct tcgcgggcaa gcccgctcct acacaggggt gatgtagggc ttgcgcgcgg
     2161 tggggccgta caggcagcaa cgatgcagcc gttttaccag gccattggct tgtcccagcc
     2221 cttccagaac aaccatcgcc gcacctcgcc gtgcgccccg gtaccgatct gccactcggg
     2281 ccgcccgcta tccagcgcaa tcaccgaaat aacccccgca tggctcgccg ccaccagcgt
     2341 ttgcccgtcg gcgctgatat ccatcgcact gatggtcgag cccaggtaat gctgccaatg
     2401 ctccgtaccc tcttcgccaa acgcccgcag gtagccatag gcatcaccca caatgtactc
     2461 gccatcgcgc gctacccccg catacaccct tgcgccatcc tgcaccagcg gggtacgcgg
     2521 gtcctggctg tagtaatcgg tatccagccc gtgcaggtct gccaccctca cggccagcgt
     2581 ggcaccgctg tagaaatggc aggcattgac gatcaactgg tcaccggcgc ggttgaacaa
     2641 ggcaaagtgt gggtattcac tgcccgggcc gatgtgcgcc agcggcttga gtcgttcgtc
     2701 cagaacaaga tgtcggctgc cttgctcacc cacgacgatc agccggccat ccgccgacac
     2761 cgcaccatgc tccatgctca gccccaggct gatgtcgtcc gggtcggtgc cctcggccag
     2821 ctcttcgaga agcttggcac tggccgtcgt tttacaacgt cgtgaccgga attgccagct
     2881 ggggcgccct ctggtaaggt tgggaagccc tgcaaacagg atgaggatcg tttcgcatga
     2941 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3001 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3061 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactccaag
     3121 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3181 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3241 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3301 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3361 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3421 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcggatg cccgacggcg
     3481 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3541 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3601 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3661 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3721 agttcttctg agcgacgatg aacatcaaaa agtttgcaaa acaagcaaca gtattaacct
     3781 ttactaccgc actgctggca ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa
     3841 agccatataa ggaaacatac ggcatttccc atattacacg ccatgatatg ctgcaaatcc
     3901 ctgaacagca aaaaaatgaa aaatatcaag tttctgaatt tgattcgtcc acaattaaaa
     3961 atatctcttc tgcaaaaggc ctggacgttt gggacagctg gccattacaa aacgctgacg
     4021 gcactgtcgc aaactatcac ggctaccaca tcgtctttgc attagccgga gatcctaaaa
     4081 atgcggatga cacatcgatt tacatgttct atcaaaaagt cggcgaaact tctattgaca
     4141 gctggaaaaa cgctggccgc gtctttaaag acagcgacaa attcgatgca aatgattcta
     4201 tcctaaaaga ccaaacacaa gaatggtcag gttcagccac atttacatct gacggaaaaa
     4261 tccgtttatt ctacactgat ttctccggta aacattacgg caaacaaaca ctgacaactg
     4321 cacaagttaa cgtatcagca tcagacagct ctttgaacat caacggtgta gaggattata
     4381 aatcaatctt tgacggtgac ggaaaaacgt atcaaaatgt acagcagttc atcgatgaag
     4441 gcaactacag ctcaggcgac aaccatacgc tgagagatcc tcactacgta gaag
//
