LOCUS       sequence-470066-        7763 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 7763)
  TITLE     Arrowsmith Lab SGC Plasmids
REFERENCE   2  (bases 1 to 7763)
  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_FL_NHis_CAvi
FEATURES             Location/Qualifiers
     source          1..7763
                     /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             3930..3974
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4027..4044
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4097..4115)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4111..4158
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4195..4650
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4282..4301)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4492..4513
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4742..5557)
                     /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(4742..5557)
                     /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     5465..5484
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5549..5568
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5679..6267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6168..6187
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6421..6438
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6453..6592
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6518..6537)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6678..6700
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6694..6885)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7694..7763,1..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 gatctgggtg ggtcagtcga aaccccacgg cgccatatca
     1561 aggttactac atacccgggg gaagagatga taggaagcta tgctgtcttg agtcctcagg
     1621 cggtgctcaa ctccgagaaa ttggcctgca tccatccact agcggagcaa gtattggtta
     1681 tgactcataa ggggcgcgca ggacggtata aggttgaacc ataccacggt cgagtgatcg
     1741 tgccttcggg aaccgcaatt ccgatcccgg attttcaggc attgtcagag tctgctacta
     1801 ttgtcttcaa tgaacgtgaa tttgttaacc ggtaccttca tcacatagcg gtgaatggtg
     1861 gagcgctgaa tactgatgaa gagtactata aggtcgttaa gtccactgaa acagattcag
     1921 agtacgtctt cgacatagac gcgaagaaat gtgtaaagaa aggagacgct ggtcctatgt
     1981 gtttggtcgg ggaactagtc gacccgccat tccacgagtt tgcatatgaa tctctcaaga
     2041 cgaggccggc agcgccccac aaagtcccca ctataggagt gtatggcgtg ccgggctcag
     2101 gcaagtccgg cataattaag tctgccgtta cgaagcggga cctggtggtt tcggcgaaga
     2161 aagaaaactg catggagatt atcaaagatg taaagaggat gcgaggaatg gacatcgccg
     2221 cacgtaccgt agactccgtt cttctcaacg gggtaaagca ctctgttgat acactttata
     2281 ttgacgaggc gtttgcttgc catgcaggaa cactattggc gctcatcgcc atagttaagc
     2341 caaaaaaagt ggtcctctgt ggcgatccca aacagtgtgg tttctttaat atgatgtgcc
     2401 taaaggtgca ctttaaccac gagatatgca cggaggtcta tcataagagt attagtcgtc
     2461 gctgtactaa aacagtcact tcaatagtct cgaccctgtt ttacgacaaa aggatgagaa
     2521 cagtaaatcc ttgcaatgat aagatcataa tcgacaccac aagcaccacc aaacctttaa
     2581 aagacgatat aatactcacg tgctttagag gttgggtaaa acaactacaa attgattata
     2641 aaaatcacga aattatgacc gcagcggcta gccaaggttt aacgcgcaag ggagtctacg
     2701 ctgtgcgata caaggttaac gaaaacccat tatacgccca gaccagcgaa catgtaaacg
     2761 tattgttaac aagaactgag aaacggatcg tctggaaaac gctggcaggc gacccttgga
     2821 taaaaaccct gacggcgtcg tacccaggta atttcacagc tacccttgaa gagtggcaag
     2881 ccgagcatga tgctattatg gccaagattc tagaaactcc agcttcgagt gacgtattcc
     2941 agaacaaggt aaatgtctgt tgggcgaaag cacttgagcc ggtacttgca acagctaata
     3001 taaccttgac gcgatcccaa tgggagacga tcccagcgtt caaagatgac aaagcctact
     3061 cgcccgaaat ggccctgaat ttcttctgta ccagattttt cggcgtggat attgattccg
     3121 gtcttttttc ggcacctacg gtacctctta cttatacaaa cgagcattgg gacaacagcc
     3181 ctggcccgaa tatgtacggg ctatgcatga gaactgccaa ggagctcgcc aggcggtatc
     3241 cgtgcatcct gaaagcggta gacactggtc gtgttgcgga tgttcgtacg gacaccatta
     3301 aagattataa ccccttgatt aatgttgtac cgttaaacag gaggctacca cactctttag
     3361 ttgttacgca ccgatatacc gggaacggcg attattctca gctagtcaca aagatgactg
     3421 ggaaaactgt cttagtcgtt ggtactccca tgaatatccc cggtaagagg gtggagacat
     3481 tgggtccaag tcctcaatgt acatacaaag ctgaactcga cttaggcatt ccagctgcct
     3541 tgggtaagta tgatattatt tttataaatg ttcggacacc ctaccgacac catcattacc
     3601 agcagtgcga ggatcacgct atccaccatt ccatgctgac gcgcaaagca gtggaccacc
     3661 taaacaaggg cggaacatgt atcgcactgg gatacgggac ggcggatcga gctaccgaaa
     3721 acataattag cgcggtggcg agatcgtttc gtttctctcg tgtgtgccaa cccaaatgtg
     3781 cctgggaaaa tacggaggta gccttcgttt tctttggtaa ggataacgga aatcatcttc
     3841 aagaccagga ccgcctctca gtagtgttaa ataacatata tcaaggaagc actcagcacg
     3901 aagcagaacg tagcagtaaa ggtggatacg gcctgaatga tatctttgaa gcgcagaaga
     3961 ttgaatggca tgaatgataa ggatccgaat tcgagctccg tcgacaagct tgcggccgca
     4021 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     4081 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     4141 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     4201 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4261 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4321 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4381 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4441 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4501 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4561 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4621 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     4681 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     4741 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     4801 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     4861 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     4921 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     4981 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     5041 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     5101 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     5161 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     5221 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     5281 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     5341 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     5401 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     5461 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     5521 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     5581 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5641 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5701 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5761 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5821 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5881 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5941 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6001 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6061 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6121 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6181 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6241 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6301 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6361 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6421 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     6481 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     6541 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     6601 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     6661 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6721 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     6781 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     6841 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     6901 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     6961 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     7021 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     7081 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     7141 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     7201 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     7261 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     7321 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     7381 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     7441 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     7501 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     7561 agtcataagt gcggcgacga tagtcatgtc ccgcgcccac cggaaggagc tgactgggtt
     7621 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     7681 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7741 atgaatcggc caacgcgcgg gga
//
