LOCUS       sequence-470059-        6404 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 6404)
  TITLE     Arrowsmith Lab SGC Plasmids
REFERENCE   2  (bases 1 to 6404)
  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: CHICKV_KY575571.1_NSP2_Pro_NHis_CAvi
FEATURES             Location/Qualifiers
     source          1..6404
                     /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             2571..2615
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2668..2685
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2738..2756)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2752..2799
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2836..3291
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2923..2942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3133..3154
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3383..4198)
                     /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(3383..4198)
                     /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     4106..4125
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4190..4209
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4320..4908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4809..4828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5062..5079
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5094..5233
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5159..5178)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5319..5341
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5335..5526)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6335..6404,1..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 tggcagggat atgtagtcat cagatgactt
     1561 tcgacacgtt tcagaataag gccaatgttt gctgggcaaa atcactggtc cctattctcg
     1621 agaccgcagg catcaagctc aacgacaggc agtggtcaca aattatccaa gcgttcaaag
     1681 aagacaaggc gtattccccc gaggtagcat taaacgagat ttgtactcgt atgtatggag
     1741 ttgacctcga ttcgggattg ttctccaagc cattagtctc tgtctattac gcagacaacc
     1801 actgggacaa cagaccagga gggaaaatgt ttggcttcaa ccccgaggcg gcgtccatcc
     1861 tggagagaaa atatcccttc accaagggta aatggaatat caacaaacaa atttgtgtta
     1921 ctacaagacg gattgaggac ttcaacccta ctactaacat aataccagcc aatcggcgac
     1981 taccacactc tctggtagcc gaacatcgac cggtcaaagg tgaacgcatg gagtggcttg
     2041 tcaacaaaat taatggacat catgtacttt tggtctccgg ctataatctt gcacttccta
     2101 caaagcgggt gacgtgggta gcaccattag gtgtaagagg agctgactat acatataatc
     2161 tagaactagg tctgccagct acattgggtc ggtatgacct cgtcgtcata aacattcata
     2221 cgccttttag gattcatcac taccagcagt gtgtggacca tgctatgaag ttacaaatgc
     2281 tcggtgggga ttctctgcga ttacttaaac ctggaggtag cttattgatc cgagcctacg
     2341 gttacgctga tcgaacctcg gaacgtgtca tttgcgtcct gggccgtaaa ttcaggagct
     2401 ccagagcact aaaaccgccc tgtgtcacgt caaatacaga aatgttcttt ctgttctcga
     2461 attttgataa cgggcgtcgt aactttacaa cgcatgtgat gaataaccag ttaaacgccg
     2521 ctttcgtggg ccaagtgaca cgggcagaat gcagcagtaa aggtggatac ggcctgaatg
     2581 atatctttga agcgcagaag attgaatggc atgaatgata aggatccgaa ttcgagctcc
     2641 gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
     2701 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2761 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2821 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2881 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2941 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3001 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3061 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3121 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3181 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3241 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3301 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3361 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3421 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3481 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3541 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3601 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3661 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3721 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3781 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3841 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3901 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3961 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     4021 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     4081 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     4141 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     4201 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     4261 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4321 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4381 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4441 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4501 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4561 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4621 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4681 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4741 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4801 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4861 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4921 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4981 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5041 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     5101 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     5161 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     5221 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5281 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5341 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5401 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5461 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5521 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5581 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5641 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5701 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5761 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5821 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5881 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5941 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     6001 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     6061 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     6121 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     6181 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgt cccgcgccca
     6241 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     6301 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     6361 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg ggga
//
