LOCUS       sequence-218346-        5377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the alpha (pcaG) and the beta (pcaH) subunits of 
            Protocatechuate 3,4-Dioxygenase from Pseudomonas putida..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5377)
  AUTHORS   Knoot CJ, Purpero VM, Lipscomb JD
  TITLE     Crystal structures of alkylperoxo and anhydride intermediates in an 
            intradiol ring-cleaving dioxygenase.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Jan 13;112(2):388-93. doi: 
            10.1073/pnas.1419118112. Epub 2014 Dec 29.
  PUBMED    25548185
REFERENCE   2  (bases 1 to 5377)
  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"; doi: 
            "10.1073/pnas.1419118112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-01-13- 13"; volume: "112"; issue: "2"; pages: "388-93"
COMMENT     Sequence Label: pVP91A-pcaHG
FEATURES             Location/Qualifiers
     source          1..5377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     84..102
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(126..140,141..146,147..163,164..170)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    146..162
                     /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    178..194
                     /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)."
     RBS             213..224
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             234..251
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1710..1728)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     protein_bind    complement(1742..1766)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      1852..1938
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2031..3113)
                     /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     3075..3094
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3114..3191)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(3236..3258)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3344..3484
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3399..3418
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3499..3516)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3670..4258)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3750..3769)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4437..5225,5226..5294))
                     /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     5057..5076
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5295..5366)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
       61 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcacctcg
      121 agaaatcata aaaaatttat ttgctttgtg agcggataac aattataata gattcaattg
      181 tgagcggata acaatttcac acagaattca ttaaagagga gaaattaacc atgcatcatc
      241 accatcacca tgcgatcgca atgcccgccc aggacaacag ccgcttcgtg atccgtgatc
      301 gcaactggca ccccaaagcc cttacgcctg actacaaaac gtccattgcc cgctcgccgc
      361 gccaggcact ggtcagcatt ccacagtcga tcagcgaaac cactggtccg aacttttccc
      421 acctgggctt cggcgcccac gaccatgacc tgctgctgaa cttcaacaac ggtggcctgc
      481 caatcggcga gcgcatcatc gtggccggcc gcgtcgtcga ccagtacggc aagcctgtgc
      541 cgaacaccct ggtggagatg tggcaagcca acgccggtgg ccgctaccgg cacaagaacg
      601 accgttacct ggcaccgctg gacccgaact ttggtggtgt cggccgttgc ctgaccgaca
      661 gcgacggcta ctacagcttc cgcaccatca agccgggccc gtacccctgg cgcaacggcc
      721 cgaacgactg gcgcccggcg cacatccact tcggcatcag cggcccgtcg attgcgacca
      781 agctgatcac ccagttgtat ttcgagggtg acccgctgat cccgatgtgc ccgatcgtca
      841 agtcgatcgc caaccctgaa gctgtacagc agttgatcgc caagctcgac atgaacaacg
      901 ccaacccgat ggactgcctg gcctaccgct ttgacatcgt gctgcgcggc cagcgcaaga
      961 cccacttcga aaactgctga ggaacccgcc atgccaatcg aactgctgcc ggaaacccct
     1021 tcgcagaccg ccggccccta cgtgcacatc ggcctggccc tggaagcggc cggcaacccg
     1081 acccgcgatc aggaaatctg gaaccgcctg gccaagccgg acgcgccagg cgagcacatt
     1141 ctgctactcg gccaggtgta tgacggtaac ggccacctgg tgcgcgactc gttcctggaa
     1201 gtgtggcagg ccgacgccaa tggcgagtat caggatgcct acaacctgga gaacgccttc
     1261 aacagcttcg gccgcaccgc caccaccttc gatgctggcg agtggacgct gcacacggtc
     1321 aagccgggtg tggtgaacaa tgctgctggc gtgccgatgg cgccgcacat caacatcagc
     1381 ctgtttgccc gtggcatcaa catccacctg cacacgcgcc tgtacttcga tgatgaagcc
     1441 caagccaacg ccaagtgccc ggtgctcaac ctgattgagc agccgcagcg gcgtgaaacc
     1501 ttgattgcca agcgttgcga agtggatggg aaaacggcgt atcgtttcga tatccgtatt
     1561 cagggggaag gcgagaccgt cttcttcgac ttctgagttt aaacgaattc gagctcggta
     1621 cccggggatc ctctagagtc gacctgcagg catgcaagct gatccggctg ctaacaaagc
     1681 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcaa actcgtttct
     1741 cgttcagctt tcttgtacaa agtggtgatg gcgcgcctgt aggacgtcga cggtaccatc
     1801 gatacgcgtt cgaagcttcg cctggggtaa tgactctcta gcttgaggca tcaaataaaa
     1861 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     1921 ctcctgagta ggacaaatcc gccctctaga ttacgtgcag tcgatgataa gctgtcaaac
     1981 atgagaattg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     2041 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2101 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     2161 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     2221 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     2281 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     2341 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     2401 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     2461 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     2521 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     2581 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     2641 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     2701 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     2761 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     2821 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     2881 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     2941 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     3001 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     3061 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     3121 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     3181 tcgatggtgt cggaacctag agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3241 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3301 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3361 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     3421 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     3481 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3541 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3601 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3661 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3721 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3781 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3841 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3901 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3961 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4021 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4081 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     4141 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4201 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4261 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4321 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4381 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagcc taggtaccaa
     4441 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4501 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4561 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4621 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4681 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4741 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4801 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4861 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4921 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4981 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5041 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5101 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5161 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5221 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5281 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5341 ctcatgagcg gatacatatt tgaatgtatt cgtacgc
//
