LOCUS       sequence-469870-        6418 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 6418)
  TITLE     Arrowsmith Lab SGC Plasmids
REFERENCE   2  (bases 1 to 6418)
  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_NHisTEV_CThromAvi
FEATURES             Location/Qualifiers
     source          1..6418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      16..471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(103..122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     313..334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(563..1378)
                     /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(563..1378)
                     /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     1286..1305
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1370..1389
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1500..2088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1989..2008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2242..2259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2274..2413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2339..2358)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2499..2521
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /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     4559..4578
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4881..4900
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4984..5003
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4984..5002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5003..5027
                     /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             5042..5064
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5074..5091
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5104..5124
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6139..6156
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6163..6207
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6217..6237
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             6266..6283
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6336..6354)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6350..6397
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
       61 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      121 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      181 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      241 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      301 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
      361 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
      421 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
      481 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
      541 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
      601 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
      661 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
      721 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
      781 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
      841 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
      901 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
      961 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     1021 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     1081 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     1141 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     1201 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     1261 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     1321 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     1381 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     1441 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1501 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1561 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1621 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1681 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1741 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1801 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1861 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1921 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1981 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2041 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2101 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2161 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2221 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     2281 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5041 ttttgtttaa ctttaagaag gagatatacc atgcatcatc atcatcatca cagcagcggc
     5101 agagaaaact tgtatttcca gggcatggcc aagattctag aaactccagc ttcgagtgac
     5161 gtattccaga acaaggtaaa tgtctgttgg gcgaaagcac ttgagccggt acttgcaaca
     5221 gctaatataa ccttgacgcg atcccaatgg gagacgatcc cagcgttcaa agatgacaaa
     5281 gcctactcgc ccgaaatggc cctgaatttc ttctgtacca gatttttcgg cgtggatatt
     5341 gattccggtc ttttttcggc acctacggta cctcttactt atacaaacga gcattgggac
     5401 aacagccctg gcccgaatat gtacgggcta tgcatgagaa ctgccaagga gctcgccagg
     5461 cggtatccgt gcatcctgaa agcggtagac actggtcgtg ttgcggatgt tcgtacggac
     5521 accattaaag attataaccc cttgattaat gttgtaccgt taaacaggag gctaccacac
     5581 tctttagttg ttacgcaccg atataccggg aacggcgatt attctcagct agtcacaaag
     5641 atgactggga aaactgtctt agtcgttggt actcccatga atatccccgg taagagggtg
     5701 gagacattgg gtccaagtcc tcaatgtaca tacaaagctg aactcgactt aggcattcca
     5761 gctgccttgg gtaagtatga tattattttt ataaatgttc ggacacccta ccgacaccat
     5821 cattaccagc agtgcgagga tcacgctatc caccattcca tgctgacgcg caaagcagtg
     5881 gaccacctaa acaagggcgg aacatgtatc gcactgggat acgggacggc ggatcgagct
     5941 accgaaaaca taattagcgc agtggcgaga tcgtttcgtt tctctcgtgt gtgccaaccc
     6001 aaatgtgcct gggaaaatac ggaggtagcc ttcgttttct ttggtaagga taacggaaat
     6061 catcttcaag accaggaccg cctctcagta gtgttaaata acatatatca aggaagcact
     6121 cagcacgaag caggccgtct agtgcccagg ggttcttcgt ccggcctcaa cgatatattt
     6181 gaggctcaaa aaatcgaatg gcacgagtaa taagcagaga acctctactt ccaatctgat
     6241 gacgaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     6301 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     6361 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatcc
//
