LOCUS       sequence-220027-        6541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SaNAS a nicotianamine synthase from Staphylcoccus aureus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6541)
  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 6541)
  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: pET15TV-L-SaNAS
FEATURES             Location/Qualifiers
     source          1..6541
                     /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             130..150
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1028..1046)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1042..1089
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1264..1269,1270..1286,1287..1292))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1319..1337)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1405..1509
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1510..1578,1579..2370)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1728..1747)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2541..3129
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3030..3049
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3283..3300
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3329..3468
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3394..3413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3554..3576
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3570..3761)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5073..6155)
                     /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     6117..6136
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6156..6233)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6439..6458
                     /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 agcggcagag aaaacttgta tttccagggc catatgaaca acttcaacaa cgagattaag
      181 ctgatcctgc agcaatacct ggagaaattt gaagcgcact atgaacgtgt tctgcaggac
      241 gatcaataca tcgaggcgct ggaaaccctg atggacgatt acagcgagtt cattctgaac
      301 ccgatctatg aacagcaatt taacgcgtgg cgtgacgttg aggaaaaggc gcagctgatt
      361 aaaagcctgc aatacatcac cgcgcagtgc gtgaagcaag ttgaggtgat tcgtgcgcgt
      421 cgtctgctgg acggtcaggc gagcaccacc ggttatttcg acaacattga acactgcatc
      481 gatgaggaat ttggtcaatg cagcattacc agcaacgata agctgctgct ggttggtagc
      541 ggcgcgtatc cgatgaccct gatccaggtt gcgaaagaga ccggtgcgag cgtgatcggc
      601 attgacatcg atccgcaagc ggtggacctg ggtcgtcgta ttgttaacgt gctggcgccg
      661 aacgaggaca ttaccatcac cgatcagaaa gttagcgaac tgaaggacat caaagatgtg
      721 acccacatca ttttcagcag caccattccg ctgaaataca gcatcctgga ggaactgtat
      781 gacctgacca acgaaaacgt ggttgtggcg atgcgtttcg gtgatggcat taaggcgatc
      841 tttaactacc cgagccagga gaccgcggag gataagtggc aatgcgtgaa caaacacatg
      901 cgtccgcagc aaatttttga catcgcgctg tataagaaag cggcgattaa agttggtatc
      961 accgatgtgt aactcgagga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1021 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1081 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
     1141 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     1201 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     1261 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     1321 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1381 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1441 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1501 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1561 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1621 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1681 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1741 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1801 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1861 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1921 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1981 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2041 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2101 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2161 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2221 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2281 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2341 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2401 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2461 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2521 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2581 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2641 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2701 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2761 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2821 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2881 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2941 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3001 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3061 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3121 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3181 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3241 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3301 gaagcgtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3361 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3421 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3481 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3541 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3601 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3661 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     3721 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     3781 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     3841 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     3901 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     3961 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4021 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4081 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4141 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4201 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     4261 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     4321 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     4381 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     4441 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     4501 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     4561 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     4621 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     4681 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     4741 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4801 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4861 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4921 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4981 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     5041 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5101 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5161 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     5221 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     5281 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     5341 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5401 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5461 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5521 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5581 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5641 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5701 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5761 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5821 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5881 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5941 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     6001 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     6061 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     6121 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6181 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     6241 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     6301 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     6361 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     6421 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     6481 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     6541 t
//
