LOCUS       sequence-431364-        4499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  deletion of calB.
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: pIAR006
FEATURES             Location/Qualifiers
     source          1..4499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      895..1483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1384..1403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1495..1511
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1495..1511
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3041..3058)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3041..3057)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3139..3933
                     /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(3193..3212)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3803..3822
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3940..4026,4027..4499,1..862)
                     /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 cggcaaacaa acactgacaa ctgcacaagt taacgtatca gcatcagaca gctctttgaa
       61 catcaacggt gtagaggatt ataaatcaat ctttgacggt gacggaaaaa cgtatcaaaa
      121 tgtacagcag ttcatcgatg aaggcaacta cagctcaggc gacaaccata cgctgagaga
      181 tcctcactac gtagaagata aaggccacaa atacttagta tttgaagcaa acactggaac
      241 tgaagatggc taccaaggcg aagaatcttt atttaacaaa gcatactatg gcaaaagcac
      301 atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc gataaaaaac gcacggctga
      361 gttagcaaac ggcgctctcg gtatgattga gctaaacgat gattacacac tgaaaaaagt
      421 gatgaaaccg ctgattgcat ctaacacagt aacagatgaa attgaacgcg cgaacgtctt
      481 taaaatgaac ggcaaatggt acctgttcac tgactcccgc ggatcaaaaa tgacgattga
      541 cggcattacg tctaacgata tttacatgct tggttatgtt tctaattctt taactggccc
      601 atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg gatcttgatc ctaacgatgt
      661 aacctttact tactcacact tcgctgtacc tcaagcgaaa ggaaacaatg tcgtgattac
      721 aagctatatg acaaacagag gattctacgc agacaaacaa tcaacgtttg cgccgagctt
      781 cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa gacagcatcc ttgaacaagg
      841 acaattaaca gttaacaaat aatcagaccc cgtagaaaag atcaaaggat cttcttgaga
      901 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
      961 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1021 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     1081 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1141 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     1201 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1261 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1321 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1381 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     1441 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcct cacacaggaa
     1501 acagctatga catgattacg aattcggcat tgtcggtgac gaaaccgagc tccagcacca
     1561 gcacctgata gacgccgcgc ttgaaggctt cttcacccaa gtgcgcagaa tgttcacgcg
     1621 tggtgctgag aaaacgtacc catgatgtca gcacgatcca ggcgttgatg gtcagtgact
     1681 cgatctgcgc cggcaccatg gccaggatgc cagcctcgac gaagccgcgg tagatcgcct
     1741 ggccctggcg caggcagcgc tcggaaaaac gccggtaacg ggcggccagt tcggcatcgc
     1801 tgtccagcag atgctccagg tcacggtgca ggaagcggta gttccacatc gccgccagca
     1861 aggccttgag gtagaagcgc ttgtcttcca cggttgccgc gcggccctgc ggcgggcgca
     1921 gaaaactgtc caccagcgct tcgtactggc tgaacaagag ggcaatgatc gcctgcttgt
     1981 tggggaagtg gtaatacagg ttgccgggcg agatttccat gtgcgcggca atgtggttgg
     2041 tactgacgct gcgttcgccc tgctggttga acagctccag gctgttctgc acgatgcgct
     2101 ctcgggtctt catgcgcggg gccatgctcg ctcccagtct gcgggccttc atcaaaggtt
     2161 tatcttacgg cgtattacag atggaatgca ccgttgaacg gcatgtaaaa attcgcacca
     2221 tggtccaaat tagagtatag gctctaggca accccggccc ggactcgaca tcaccgatat
     2281 cggatcgcac catgcaccgc cgcgacctgc tgcgtttcag cctcggcgcc agtgcttttc
     2341 tggcgggtgc cagcctggtg gggtgcagcg tgcaaaccgc cgcgaccggc tatcaggtac
     2401 ttcgtgacga cgacctgccc ctgctgcgtg cgctgatccc ggtggtcctg gccggtacac
     2461 aggccaccga tgcgccggtg ttgcgcagcc tcgacaacaa gctggcgtcg ttgtcgccgg
     2521 aaatgctcaa gctcacccgg caattgttcg acgtgctcag cctgccgctg actcgcggcc
     2581 cgctgaccgg cgtctggggc agctggggcc aagctggcgc cacgcaggtc acggcgttcc
     2641 tgcaacgctg gcaggacagc tcgctgaacc tgctacgcat ggggcacgcc tcgttgctgc
     2701 aactgctgca aatggcctgg tacgaacgcc ccgaatcctg ggcagactgc ggttacccgg
     2761 gcccaccgaa aatctgaaaa caacaagagc atcgccaatg cctgtccccg acccgtttcg
     2821 ccaaggcctc gaacgcggct ggatcaccca cgacgcctcg cgcctggagc acgacctgtt
     2881 tctggaggcc gacattgccg tgatcggcag tggtgccggc ggcgccacca gtgcgcagat
     2941 gctcagcgct gccggtttca aggtactgct gatcgaagaa ggcccactga agaccagcag
     3001 cgatttccac ctgctggaga gcgaagccta caagcttggc actggccgtc gttttacaac
     3061 gtcgtgaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaaca
     3121 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3181 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3241 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3301 ggtgccctga atgaactcca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     3361 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3421 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3481 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3541 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3601 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3661 aaggcgcgga tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3721 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     3781 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     3841 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3901 gccttctatc gccttcttga cgagttcttc tgagcgacga tgaacatcaa aaagtttgca
     3961 aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc aactcaagcg
     4021 tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc ccatattaca
     4081 cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca agtttctgaa
     4141 tttgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt ttgggacagc
     4201 tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca catcgtcttt
     4261 gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt ctatcaaaaa
     4321 gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa agacagcgac
     4381 aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc aggttcagcc
     4441 acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg taaacatta
//
