LOCUS       sequence-220085-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PaODH and opine dehydrogenase from Pseudomonas aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6591)
  AUTHORS   McFarlane JS, Lamb AL
  TITLE     Biosynthesis of an Opine Metallophore by Pseudomonas aeruginosa.
  JOURNAL   Biochemistry. 2017 Nov 14;56(45):5967-5971. doi: 
            10.1021/acs.biochem.7b00804. Epub 2017 Nov 3.
  PUBMED    29091735
REFERENCE   2  (bases 1 to 6591)
  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: 
            "10.1021/acs.biochem.7b00804"; journalName: "Biochemistry"; date: 
            "2017-11-14- 14"; volume: "56"; issue: "45"; pages: "5967-5971"
COMMENT     Sequence Label: pET28b-PaODH
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1456..1473
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1526..1544)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1540..1587
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1624..2079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1711..1730)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1921..1942
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2171..2986)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2171..2986)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2894..2913
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2978..2997
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3108..3696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3597..3616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3850..3867
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3882..4021
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3947..3966)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4107..4129
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4123..4314)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5123..6205)
                     /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     6167..6186
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6206..6283)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6489..6508
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg aacgcggcgg acgagagcct gggtaacgtg
      181 ctgctggttg gtctgggtgc ggtggcgatc caggttgcgc tggatctgcg tcgtcatggt
      241 gcgggccgtc tgggtgcgct gaaccacccg ggtcgtcgta gccaacgtat tgcggaggcg
      301 ctggcgcgtg gtgcgtgcct gcaactggaa ggccagggcc aacatcgttg gctgagcggt
      361 aacgcggcgc tggacgtttt ccaccaggat ccggcggagc tgcgtgacga ttggcaaacc
      421 ctggtgctgt gcgttccggc ggacagctac ctggatgtgg ttcgtggtct gccgtgggaa
      481 cgtctgggtg gcgtgcgtac cctgctgctg gttagcgcgt ttatcggtgc gaacctgctg
      541 gtgcgtagcg cgctgccggc gggttgccag gcgaccgttc tgagcctgag cagctactat
      601 gcggcgacca aagtgattga cgaaacccag ccgctgcgtg cgctgaccaa ggcggtgaaa
      661 cgtcgtgttt acctgggcag cagccgtccg gattgcccgg cgcgtgagac ctggcgtcgt
      721 gtgctggcgg gtagcggcgt tgaagtggtt ccgctggcga ccccggaggc ggcggaaggt
      781 cgtaacgtga ccacctatgt tcacagcccg ttctttctgg gcgagttcgc gctggcgcgt
      841 atcctgagcg aacagggtcc gccgggcttt atgtacaaac tgtatccgga aggtccgatt
      901 accccgggtg cgattggtgc gatgcgtcgt ctgtggtgcg agctgagcga actgctgcgt
      961 cgtatgggcg cggaaccgct gaacctgctg cgtttcctga acgacgataa ctatccggtt
     1021 cacgagacca tgctgccgcg tgcgagcatt gacggttttg cggaagcggg cgcggagcgt
     1081 caggaatacc tgctgttcgt gcgttatgcg gcgctgctgg ttgacccgtt tagcccggcg
     1141 gatgagcaag gtcgtcactt cgattttagc gcggtgccgt tccgtcgtgt tagccgtgac
     1201 gaggatggtc tgtggcgtct gccgcgtgtt ccgctggaag actaccgtaa actggcgctg
     1261 attgtggcgc tggcggcgca ctttgatctg gcgatgccgc aggcgcgtag cctgctggcg
     1321 agctatgaaa acgctgtgag ccgttttatt gactgccaag gtgcgagcca atgccacccg
     1381 agcctgtatc cgattgacag ccgtccggcg gcggatgcga tttatcgtca atggtgcagc
     1441 acctgctaac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1501 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1561 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1621 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1681 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1741 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1801 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1861 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1921 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1981 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2041 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     2101 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2161 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     2221 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     2281 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     2341 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     2401 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     2461 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2521 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     2581 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     2641 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     2701 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2761 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2821 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2881 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2941 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3001 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     3061 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3121 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3181 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3241 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3301 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3361 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3421 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3481 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3541 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3601 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3661 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3721 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3781 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3841 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3901 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3961 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4021 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4081 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4141 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4201 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4261 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4321 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4381 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4441 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4501 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4561 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4621 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4681 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4741 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4801 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4861 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4921 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4981 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     5041 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5101 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5161 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     5221 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     5281 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     5341 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     5401 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5461 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5521 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5581 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5641 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5701 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5761 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5821 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5881 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5941 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     6001 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     6061 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     6121 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     6181 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     6241 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6301 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     6361 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     6421 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     6481 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     6541 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
