LOCUS       sequence-470057-        5927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination. May not contain 
            entire coding region of gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5927)
  TITLE     Arrowsmith Lab SGC Plasmids
REFERENCE   2  (bases 1 to 5927)
  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"
COMMENT     Sequence Label: SARS2nsp3_MacroD_ECBio2
FEATURES             Location/Qualifiers
     source          1..5927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     975..994
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1014..1091)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1297..1316
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1400..1419
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1400..1418
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1419..1443
                     /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             1458..1480
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1491..1508
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1533..1553
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2058..2075
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2094..2138
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2191..2208
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2261..2279)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2275..2322
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2359..2814
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2446..2465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2656..2677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2906..3721)
                     /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(2906..3721)
                     /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     3629..3648
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3713..3732
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3843..4431
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4332..4351
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4585..4602
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4617..4756
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4682..4701)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4842..4864
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4858..5049)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5858..5927,1..1013))
                     /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
                     EACKAAVHNLLAQRVSGLIINYQLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
       61 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
      121 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
      181 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
      241 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
      301 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
      361 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
      421 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
      481 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
      541 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
      601 gcttccacag caatggcatc ctggtcatcc agctgatagt taatgatcag cccactgacg
      661 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
      721 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
      781 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
      841 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
      901 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
      961 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     1021 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     1081 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     1141 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     1201 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     1261 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     1321 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     1381 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     1441 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg caccatcatc
     1501 atcatcattc ttctggtgta gatctgggta ccgagaacct gtacttccaa tccatggtga
     1561 acagcttctc cggctacctg aagctgaccg acaacgtgta catcaagaac gctgacatcg
     1621 tggaggaagc caagaaggtg aaaccaaccg tggtggtgaa cgccgctaac gtgtacctga
     1681 agcacggcgg aggtgtggct ggagctctga acaaggctac caacaacgcc atgcaggtgg
     1741 aatccgacga ctacatcgct accaacggtc ctctgaaggt gggcggatcc tgcgtgctgt
     1801 ctggccacaa cctggccaag cactgcctgc acgtggtggg accaaacgtg aacaagggag
     1861 aggacatcca gctgctgaag agcgcctacg aaaacttcaa ccagcacgag gtgctgctgg
     1921 ctccactgct gtctgccgga atcttcggtg ctgatcccat ccacagcctg cgcgtgtgcg
     1981 tggacaccgt gaggaccaac gtgtacctgg ccgtgttcga caagaacctg tacgacaagc
     2041 tggtgagctc cttcctgctt gtgcctcgtg gaagcagcag taaaggtgga tacggcctga
     2101 atgatatctt tgaagcgcag aagattgaat ggcatgaatg ataaggatcc gaattcgagc
     2161 tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
     2221 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     2281 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     2341 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2401 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2461 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2521 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2581 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2641 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2701 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2761 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2821 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2881 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     2941 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     3001 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     3061 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     3121 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     3181 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     3241 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     3301 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     3361 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     3421 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     3481 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     3541 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     3601 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     3661 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     3721 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     3781 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3841 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3901 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3961 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4021 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4081 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4141 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4201 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4261 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4321 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4381 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4441 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4501 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4561 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4621 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     4681 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4741 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4801 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4861 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4921 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4981 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     5041 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     5101 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     5161 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     5221 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     5281 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     5341 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     5401 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     5461 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     5521 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     5581 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5641 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5701 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgtcccgcgc
     5761 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5821 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5881 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcgggga
//
