LOCUS       sequence-469864-        6098 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 6098)
  TITLE     Arrowsmith Lab SGC Plasmids
REFERENCE   2  (bases 1 to 6098)
  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: Denv4_KJ160504.1_NS2B
FEATURES             Location/Qualifiers
     source          1..6098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     206..225
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     309..328
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        309..327
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    328..352
                     /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             367..389
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             408..425
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             435..452
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             453..497
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1271..1288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1341..1359)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1355..1402
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1439..1894
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1526..1545)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1736..1757
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1986..2801)
                     /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(1986..2801)
                     /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     2709..2728
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2793..2812
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2923..3511
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3412..3431
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3665..3682
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3697..3836
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3762..3781)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3922..3944
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3938..4129)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4938..6020)
                     /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
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5982..6001
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6021..6098)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
       61 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
      121 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
      181 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      241 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      301 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      361 aataattttg tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc
      421 atcacagcag cggcctggtt ccgcgtggta gtgggctcaa cgacattttt gaagcccaaa
      481 agatcgaatg gcatgaggga tccgctggtg gatcgggtgc tgatctgtct cttgaaaaag
      541 cggccaacgt acagtgggat gaaatggcag acataactgg aagttcccct ataatcgagg
      601 tgaaacagga tgaggatggt tctttcagca ttcgtgatgt tgaagaaacc aatatgattg
      661 gcggcggagg ctccgggggc ggtggttcag gggccctgtg ggatgtaccg tcaccagcgg
      721 ccacacaaaa agcggcgctc tcggaggggg tctaccggat tatgcagcgt gggctttttg
      781 gtaaaaccca agtcggtgtt ggtattcata tggaaggtgt ttttcatacc atgtggcatg
      841 tgacgagggg tagcgttatt tgtcatgaaa ccggtcgttt ggagccgagc tgggcagatg
      901 ttcgtaatga tatgattagc tatggtggcg gttggcgtct gggtgataaa tgggacaaag
      961 aagaggacgt acaagtgtta gccatcgagc ccggcaaaaa tccgaaacat gttcaaacaa
     1021 agccaggact tttcaaaact ttaacaggtg aaattggtgc cgtaacactg gattttaagc
     1081 ccggtacatc cggaagcccg attatcaatc ggaaaggtaa agttatcggg ctttacggta
     1141 atggtgtagt aactaagtca ggcgattatg ttagcgcaat tacccaggca gaacgcatcg
     1201 gggagccgga ttatgaagtt gatgaggaca tcttcagaaa gtgatgacga agcttgcggc
     1261 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     1321 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1381 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     1441 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1501 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1561 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1621 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1681 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1741 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1801 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1861 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     1921 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     1981 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2041 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2101 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2161 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2221 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2281 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2341 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2401 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2461 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2521 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2581 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2641 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2701 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2761 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     2821 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     2881 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2941 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3001 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3061 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3121 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3181 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3241 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3301 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3361 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3421 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3481 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3541 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3601 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3661 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3721 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3781 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3841 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3901 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3961 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     4021 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4081 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4141 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4201 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4261 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4321 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4381 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4441 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4501 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4561 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4621 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4681 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4741 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4801 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4861 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4921 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4981 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5041 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5101 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5161 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     5221 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     5281 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     5341 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5401 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5461 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5521 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5581 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5641 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5701 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5761 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5821 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5881 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5941 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6001 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6061 gccataccgc gaaaggtttt gcgccattcg atggtgtc
//
