LOCUS       sequence-470065-        6395 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 6395)
  TITLE     Arrowsmith Lab SGC Plasmids
REFERENCE   2  (bases 1 to 6395)
  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: EEEV_KP282670.1_NSP2_Pro_NHis_CAvi
FEATURES             Location/Qualifiers
     source          1..6395
                     /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             2562..2606
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2659..2676
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2729..2747)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2743..2790
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2827..3282
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2914..2933)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3124..3145
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3374..4189)
                     /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(3374..4189)
                     /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     4097..4116
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4181..4200
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4311..4899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4800..4819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5053..5070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5085..5224
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5150..5169)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5310..5332
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5326..5517)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6326..6395,1..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 tggccaagat tctagaaact ccagcttcga
     1561 gtgacgtatt ccagaacaag gtaaatgtct gttgggcgaa agcacttgag ccggtacttg
     1621 caacagctaa tataaccttg acgcgatccc aatgggagac gatcccagcg ttcaaagatg
     1681 acaaagccta ctcgcccgaa atggccctga atttcttctg taccagattt ttcggcgtgg
     1741 atattgattc cggtcttttt tcggcaccta cggtacctct tacttataca aacgagcatt
     1801 gggacaacag ccctggcccg aatatgtacg ggctatgcat gagaactgcc aaggagctcg
     1861 ccaggcggta tccgtgcatc ctgaaagcgg tagacactgg tcgtgttgcg gatgttcgta
     1921 cggacaccat taaagattat aaccccttga ttaatgttgt accgttaaac aggaggctac
     1981 cacactcttt agttgttacg caccgatata ccgggaacgg cgattattct cagctagtca
     2041 caaagatgac tgggaaaact gtcttagtcg ttggtactcc catgaatatc cccggtaaga
     2101 gggtggagac attgggtcca agtcctcaat gtacatacaa agctgaactc gacttaggca
     2161 ttccagctgc cttgggtaag tatgatatta tttttataaa tgttcggaca ccctaccgac
     2221 accatcatta ccagcagtgc gaggatcacg ctatccacca ttccatgctg acgcgcaaag
     2281 cagtggacca cctaaacaag ggcggaacat gtatcgcact gggatacggg acggcggatc
     2341 gagctaccga aaacataatt agcgcagtgg cgagatcgtt tcgtttctct cgtgtgtgcc
     2401 aacccaaatg tgcctgggaa aatacggagg tagccttcgt tttctttggt aaggataacg
     2461 gaaatcatct tcaagaccag gaccgcctct cagtagtgtt aaataacata tatcaaggaa
     2521 gcactcagca cgaagcagaa cgtagcagta aaggtggata cggcctgaat gatatctttg
     2581 aagcgcagaa gattgaatgg catgaatgat aaggatccga attcgagctc cgtcgacaag
     2641 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     2701 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2761 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     2821 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2881 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2941 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3001 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3061 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3121 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3181 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3241 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3301 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3361 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     3421 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3481 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3541 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3601 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     3661 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     3721 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     3781 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3841 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     3901 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     3961 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     4021 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     4081 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     4141 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     4201 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     4261 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4321 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4381 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4441 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4501 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4561 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4621 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4681 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4741 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4801 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4861 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4921 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4981 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5041 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     5101 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     5161 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5221 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5281 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5341 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5401 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5461 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5521 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5581 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5641 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5701 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5761 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5821 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5881 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     5941 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     6001 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     6061 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     6121 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     6181 gataaagaag acagtcataa gtgcggcgac gatagtcatg tcccgcgccc accggaagga
     6241 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6301 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6361 ccagctgcat taatgaatcg gccaacgcgc gggga
//
