LOCUS       sequence-437832-        5083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For recombinant expression of vanA (PP_3736 from Pseudomonas putida)
            with a C-terminal His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5083)
  AUTHORS   Bleem AC, Kuatsjah E, Johnsen J, Mohamed ET, Alexander WG, 
            Kellermyer ZA, Carroll AL, Rossi R, Schlander IB, Peabody V GL, Guss
            AM, Feist AM, Beckham GT
  TITLE     Evolution and engineering of pathways for aromatic O-demethylation 
            in Pseudomonas putida KT2440.
  JOURNAL   Metab Eng. 2024 Jul;84:145-157. doi: 10.1016/j.ymben.2024.06.009. 
            Epub 2024 Jun 25.
  PUBMED    38936762
REFERENCE   2  (bases 1 to 5083)
  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: "Metab 
            Eng."; date: "2024-07"; volume: "84:145-157. doi"; pages: " 
            10.1016/j.ymben.2024.06.009. Epub 2024 Jun 25"
COMMENT     Sequence Label: pSN95
FEATURES             Location/Qualifiers
     source          1..5083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1416..1432
                     /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)."
     promoter        join(1457..1471,1472..1477,1478..1494,1495..1501)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1477..1493
                     /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)."
     protein_bind    1507..1526
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     RBS             1575..1583
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             2657..2674
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2880..2899)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2905..2924)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2966..3057
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3058..3126,3127..3918)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3276..3295)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             4001..5083
                     /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     complement(4020..4039)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc
     1441 ttcatgcaca gtgaaatcat gaaaaattta tttgctttgt gagcggataa caattataat
     1501 atgtggaatt gtgagcgctc acaattccac aacggtttcc ctctagaaat aattttgttt
     1561 aacttttcga gacctaagga ggtaaaaaat gtatccaaag aatacctggt atgttgcgtg
     1621 tacgccagat gaaattgcta ctaagccgct gggccgtcaa atctgtggcg agaaaatcgt
     1681 tttctaccgt gcacgtgaga atcaagtcgc ggccgttgaa gatttctgcc cgcatcgcgg
     1741 tgcgccgctg agcctgggtt acgtggaaga tggcaacctg gtttgcggtt accacggcct
     1801 ggtcatgggc tgcgatggta agaccgtgag catgccgggt caacgtgtcc gcggttttcc
     1861 gtgcaataaa accttcgcag ccgtggaacg ttacggtttt atctgggtct ggccgggtga
     1921 ccaagcgcag gcagacccgg ctctgatccc gcacctggag tgggcagtgt ccgatgagtg
     1981 ggcgtacggt ggcggcctgt tccatattgg ttgcgactac cgcttgatga tcgacaacct
     2041 gatggacttg acccacgaaa cgtatgttca cgccagcagc attggtcaga aagaaatcga
     2101 cgaagcgccg ccggttacga cggtcaccgg cgacgaagtt gtgactgccc gtcacatgga
     2161 aaacattatg gcgcctccgt tctggcgtat ggcactgcgc ggtaatggtc tggcggacga
     2221 cgtgccggtg gaccgttggc agatttgtcg ctttacccct ccgtcgcacg ttttgattga
     2281 ggtcggcgtc gcccatgctg gtaagggtgg ctatcacgct gaggcacaac ataaagcgag
     2341 ctccatcgtt gtggatttca tcaccccgga gagcgacacc tctatttggt acttttgggg
     2401 catggcgcgc aactttgcag cacacgatca gaccctgacg gataatattc gcgagggtca
     2461 gggtaagatc ttcagcgaag atctggaaat gctggagcgt cagcaacaaa acctgttagc
     2521 gcatccggag cgtaacctcc tgaaactgaa cattgatgcc ggtggcgtgc agagccgtaa
     2581 ggtactggag cgcatcattg cccaagagcg tgcgccgcag ccgcagctga ttgcgacgag
     2641 cgcgaatccg gcgggtcatc accaccacca ccattgaggt ctcaccccaa gggcgacacc
     2701 ccataattag cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg
     2761 cctggcagtt ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt
     2821 tcacttctga gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac
     2881 aaggggtgtt atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt
     2941 taattggttg taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3001 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg
     3061 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3121 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3181 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3241 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3301 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3361 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3421 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3481 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3541 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3601 gtagcgatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3661 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3721 gcccttccgg ctggctggtt tattgctgat aaatccggag ccggtgagcg tggttctcgc
     3781 ggtatcatcg cagcgctggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3841 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3901 ctgattaagc attggtaagc ggcgcgccat cgaatggcgc aaaacctttc gcggtatggc
     3961 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat atgaaaccag taacgttata
     4021 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     4081 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     4141 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     4201 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     4261 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     4321 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     4381 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     4441 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aggacggtac
     4501 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     4561 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     4621 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     4681 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     4741 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     4801 tatctcggta gtgggatacg acgataccga agatagctca tgttatatcc cgccgttaac
     4861 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     4921 ctctcagggc caggcggtga agggcaatca gctgttgcca gtctcactgg tgaaaagaaa
     4981 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5041 gcagctggca cgacaggttt cccgactgga aagcgggcag tga
//
