LOCUS       sequence-462634-        6440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Staphylococcus aureus D-alanine D-alanine under T7 with 
            HIS tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6440)
  AUTHORS   Beckham J
  TITLE     Josh Beckham Lab plasmids 2024
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6440)
  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            "Unpublished"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: SaDalaDala
FEATURES             Location/Qualifiers
     source          1..6440
                     /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             148..180
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1301..1318
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1371..1389)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1385..1432
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1469..1924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1556..1575)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1766..1787
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2017..2832)
                     /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(2017..2832)
                     /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     2740..2759
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2824..2843
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2954..3542
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3443..3462
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3696..3713
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3728..3870
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3796..3815)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3956..3978
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3972..4163)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4972..6054)
                     /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     6016..6035
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6055..6132)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6338..6357
                     /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 gctagcatga ctggtggaca gcaaatgggt
      181 cgcggatccg aattcatgac caaagagaac atctgcatcg tttttggtgg taaaagcgcg
      241 gaacacgaag tttctatcct gaccgcgcag aatgttctga acgcgatcga caaagacaaa
      301 taccacgttg acatcatcta catcacgaac gacggtgact ggcgtaaaca aaacaacatc
      361 accaccgaaa tcaaaagcac cgacgaactg cacctggaaa acggtgaagc cctcgagatc
      421 agccagctcc tgaaagaatc ttcttctggt cagccgtacg acgcggtttt cccgctgctg
      481 cacggtccga acggcgagga cggcaccatc cagggtctct tcgaggtact cgacgttccg
      541 tacgttggta acggtgtcct gtctgcggcg tcttctatgg acaaactggt tatgaagcaa
      601 ctgtttgaac accgtggtct gccacagctg ccgtacatct ctttcctgcg tagcgaatac
      661 gagaaatacg aacataacat cctgaagctg gttaacgaca agctgaacta ccctgttttc
      721 gtaaaaccgg cgaacctggg tagctctgtt ggtatctcta aatgcaacaa cgaagcggaa
      781 ctcaaagaag gtatcaaaga ggcgtttcag ttcgaccgca aactcgttat cgagcagggt
      841 gttaacgccc gtgaaatcga agttgcggtt ctgggtaacg actatccgga ggcaacctgg
      901 ccgggtgaag ttgttaaaga cgttgcgttc tacgactaca aatctaaata caaggacggt
      961 aaagttcaac tgcagattcc ggcggacctg gacgaagatg tgcagctgac cctgcgcaat
     1021 atggcgctcg aagcgtttaa agcgaccgac tgctctggtc tggttcgtgc ggacttcttc
     1081 gttacggaag acaaccagat ttacattaac gaaaccaatg cgatgccggg tttcaccgcg
     1141 ttctctatgt acccgaaact gtgggagaat atgggcctgt cttacccgga actgatcacc
     1201 aaactcattg aactggcgaa agaacgccac caggacaagc agaaaaacaa gtataagatc
     1261 gactaagagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg
     1321 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1381 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1441 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1501 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1561 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1621 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1681 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1741 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1801 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1861 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     1921 atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1981 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     2041 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     2101 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     2161 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     2221 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     2281 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     2341 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     2401 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     2461 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     2521 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     2581 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     2641 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     2701 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     2761 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     2821 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     2881 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2941 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3001 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3061 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3121 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3181 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3241 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3301 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3361 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3421 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3481 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3541 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3601 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3661 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3721 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     3781 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3841 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3901 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3961 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4021 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4081 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4141 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4201 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4261 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4321 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4381 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4441 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4501 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4561 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4621 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4681 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4741 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4801 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4861 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4921 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4981 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5041 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5101 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     5161 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5221 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     5281 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5341 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5401 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5461 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5521 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5581 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5641 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5701 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5761 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5821 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5881 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5941 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     6001 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     6061 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     6121 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6181 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     6241 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6301 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     6361 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     6421 agatctcgat cccgcgaaat
//
