LOCUS       sequence-469871-        5915 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 5915)
  TITLE     Arrowsmith Lab SGC Plasmids
REFERENCE   2  (bases 1 to 5915)
  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_EC
FEATURES             Location/Qualifiers
     source          1..5915
                     /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             2082..2126
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2179..2196
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2249..2267)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2263..2310
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2347..2802
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2434..2453)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2644..2665
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2894..3709)
                     /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(2894..3709)
                     /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     3617..3636
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3701..3720
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3831..4419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4320..4339
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4573..4590
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4605..4744
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4670..4689)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4830..4852
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4846..5037)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5846..5915,1..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 cttcctgtga tgaagcagta aaggtggata cggcctgaat gatatctttg
     2101 aagcgcagaa gattgaatgg catgaatgat aaggatccga attcgagctc cgtcgacaag
     2161 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     2221 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2281 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     2341 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2401 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2461 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2521 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2581 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2641 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2701 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2761 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2821 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2881 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     2941 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3001 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3061 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3121 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     3181 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     3241 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     3301 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3361 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     3421 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     3481 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     3541 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     3601 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     3661 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     3721 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     3781 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3841 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3901 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3961 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4021 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4081 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4141 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4201 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4261 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4321 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4381 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4441 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4501 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4561 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4621 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     4681 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4741 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4801 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4861 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     4921 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     4981 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5041 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5101 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5161 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5221 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5281 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5341 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5401 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     5461 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     5521 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     5581 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     5641 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5701 gataaagaag acagtcataa gtgcggcgac gatagtcatg tcccgcgccc accggaagga
     5761 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5821 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5881 ccagctgcat taatgaatcg gccaacgcgc gggga
//
