LOCUS       sequence-462635-        5832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Staphylococcus aureus Dihydrofolate reductase under T7 
            with HIS tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5832)
  AUTHORS   Beckham J
  TITLE     Josh Beckham Lab plasmids 2024
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5832)
  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: 
            "Unpublished"
COMMENT     Sequence Label: SaDHFR-pNIC-Bsa4
FEATURES             Location/Qualifiers
     source          1..5832
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             697..714
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(767..785)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      781..828
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      865..1320
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(952..971)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1162..1183
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1412..2227)
                     /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(1412..2227)
                     /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     2135..2154
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2219..2238
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2349..2937
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2838..2857
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3091..3108
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3123..3262
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3188..3207)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3348..3370
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3364..3555)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4364..5446)
                     /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     5408..5427
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5447..5524)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5730..5749
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatgacc ctgtctatcc tggttgcgca
      181 cgacctgcag cgtgttatcg gtttcgaaaa ccagctgccg tggcacctgc cgaacgacct
      241 gaaacacgtt aaaaaactgt ctaccggtca caccctggtt atgggtcgta aaaccttcga
      301 atctatcggt aaaccgctgc cgaaccgtcg taacgttgtt ctgacctctg acacctcttt
      361 caacgttgaa ggtgttgacg ttatccactc tatcgaagac atctaccagc tgccgggtca
      421 cgttttcatc ttcggtggtc agaccctgtt cgaagaaatg atcgacaaag ttgacgacat
      481 gtacatcacc gttatcgaag gtaaattccg tggtgacacc ttcttcccgc cgtacacctt
      541 cgaagactgg gaagttgcgt cttctgttga aggtaaactg gacgaaaaaa acaccatccc
      601 gcacaccttc ctgcacctga tccgtaaaaa ataacagtaa aggtggatac ggatccgaat
      661 tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
      721 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      781 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
      841 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      901 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      961 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1021 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1081 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1141 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1201 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1261 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1321 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1381 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     1441 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1501 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1561 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1621 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     1681 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     1741 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     1801 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     1861 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     1921 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     1981 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2041 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     2101 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     2161 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     2221 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     2281 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2341 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2401 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2461 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2521 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2581 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2641 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2701 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2761 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2821 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2881 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2941 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3001 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3061 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3121 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3181 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3241 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3301 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3361 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3421 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3481 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3541 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3601 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3661 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3721 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3781 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     3841 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     3901 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     3961 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4021 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4081 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     4141 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     4201 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     4261 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     4321 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     4381 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4441 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     4501 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     4561 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     4621 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     4681 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     4741 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     4801 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     4861 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     4921 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     4981 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5041 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     5101 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     5161 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     5221 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     5281 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     5341 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     5401 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     5461 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     5521 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     5581 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     5641 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     5701 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     5761 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     5821 atcccgcgaa at
//
