LOCUS       sequence-469863-        6773 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 6773)
  TITLE     Arrowsmith Lab SGC Plasmids
REFERENCE   2  (bases 1 to 6773)
  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_Hel_NHisCAvi
FEATURES             Location/Qualifiers
     source          1..6773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(79..1161)
                     /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"
     primer_bind     1123..1142
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1162..1239)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1445..1464
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1548..1567
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1548..1566
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1567..1591
                     /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             1606..1628
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1639..1656
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3088..3132
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             3185..3202
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3255..3273)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3269..3316
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3353..3808
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3440..3459)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3650..3671
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3900..4715)
                     /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(3900..4715)
                     /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     4623..4642
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4707..4726
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4837..5425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5326..5345
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5579..5596
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5611..5750
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5676..5695)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5836..5858
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5852..6043)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
       61 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      121 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
      181 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
      241 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
      301 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
      361 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
      421 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
      481 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
      541 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
      601 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
      661 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
      721 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
      781 ctgatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
      841 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
      901 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
      961 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     1021 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     1081 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     1141 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     1201 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     1261 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     1321 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     1381 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     1441 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     1501 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     1561 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     1621 gaaggagata tacatatgca ccatcatcat catcattctt ctggtgtaga tctgggtggg
     1681 tcagtcgaaa ccccacggcg ccatatcaag gttactacat acccggggga agagatgata
     1741 ggaagctatg ctgtcttgag tcctcaggcg gtgctcaact ccgagaaatt ggcctgcatc
     1801 catccactag cggagcaagt attggttatg actcataagg ggcgcgcagg acggtataag
     1861 gttgaaccat accacggtcg agtgatcgtg ccttcgggaa ccgcaattcc gatcccggat
     1921 tttcaggcat tgtcagagtc tgctactatt gtcttcaatg aacgtgaatt tgttaaccgg
     1981 taccttcatc acatagcggt gaatggtgga gcgctgaata ctgatgaaga gtactataag
     2041 gtcgttaagt ccactgaaac agattcagag tacgtcttcg acatagacgc gaagaaatgt
     2101 gtaaagaaag gagacgctgg tcctatgtgt ttggtcgggg aactagtcga cccgccattc
     2161 cacgagtttg catatgaatc tctcaagacg aggccggcag cgccccacaa agtccccact
     2221 ataggagtgt atggcgtgcc gggctcaggc aagtccggca taattaagtc tgccgttacg
     2281 aagcgggacc tggtggtttc ggcgaagaaa gaaaactgca tggagattat caaagatgta
     2341 aagaggatgc gaggaatgga catcgccgca cgtaccgtag actccgttct tctcaacggg
     2401 gtaaagcact ctgttgatac actttatatt gacgaggcgt ttgcttgcca tgcaggaaca
     2461 ctattggcgc tcatcgccat agttaagcca aaaaaagtgg tcctctgtgg cgatcccaaa
     2521 cagtgtggtt tctttaatat gatgtgccta aaggtgcact ttaaccacga gatatgcacg
     2581 gaggtctatc ataagagtat tagtcgtcgc tgtactaaaa cagtcacttc aatagtctcg
     2641 accctgtttt acgacaaaag gatgagaaca gtaaatcctt gcaatgataa gatcataatc
     2701 gacaccacaa gcaccaccaa acctttaaaa gacgatataa tactcacgtg ctttagaggt
     2761 tgggtaaaac aactacaaat tgattataaa aatcacgaaa ttatgaccgc agcggctagc
     2821 caaggtttaa cgcgcaaggg agtctacgct gtgcgataca aggttaacga aaacccatta
     2881 tacgcccaga ccagcgaaca tgtaaacgta ttgttaacaa gaactgagaa acggatcgtc
     2941 tggaaaacgc tggcaggcga cccttggata aaaaccctga cggcgtcgta cccaggtaat
     3001 ttcacagcta cccttgaaga gtggcaagcc gagcatgatg ctattatggc caagattcta
     3061 gaaactccaa gcagtaaagg tggatacggc ctgaatgata tctttgaagc gcagaagatt
     3121 gaatggcatg aatgataagg atccgaattc gagctccgtc gacaagcttg cggccgcact
     3181 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     3241 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     3301 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     3361 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3421 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3481 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3541 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3601 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3661 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3721 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3781 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     3841 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     3901 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     3961 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     4021 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     4081 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     4141 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     4201 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     4261 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     4321 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     4381 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     4441 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     4501 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     4561 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     4621 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     4681 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     4741 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4801 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4861 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4921 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4981 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5041 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5101 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5161 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5221 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5281 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5341 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5401 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5461 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5521 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5581 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     5641 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5701 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     5761 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5821 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5881 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     5941 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     6001 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     6061 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     6121 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     6181 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     6241 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     6301 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     6361 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     6421 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     6481 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     6541 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     6601 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     6661 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     6721 tcataagtgc ggcgacgata gtcatgtccc gcgcccaccg gaaggagctg act
//
