LOCUS       sequence-212328-        5967 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5967)
  TITLE     SGC Oxford April 2018 plasmids
REFERENCE   2  (bases 1 to 5967)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: RAC1 (RAC1A-c007)
FEATURES             Location/Qualifiers
     source          1..5967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     150..169
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     253..272
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        253..271
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    272..296
                     /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             311..333
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             359..382
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             419..442
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             467..487
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1084..1101
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1154..1172)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1168..1215
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1252..1707
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1339..1358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1549..1570
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1799..2614)
                     /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(1799..2614)
                     /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     2522..2541
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2606..2625
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2736..3324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3225..3244
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3478..3495
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3510..3649
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3575..3594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3735..3757
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3751..3942)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4751..5833)
                     /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     5795..5814
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5834..5911)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
       61 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
      121 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      181 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
      241 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      301 tagaaataat tttgtttaac tttaagaagg agatatacat atgtcttctg gtgcgagctg
      361 gagtcaccca caattcgaga aaggtggtgg ctctggtggc ggttcaggcg gagccgcctg
      421 gtcccatcca cagtttgaaa agggttctgg tgtagatctg ggtaccgaga acctgtactt
      481 ccaatccatg caggccatca agtgtgtggt ggtgggagac ggagctgtag gtaaaacttg
      541 cctactgatc agttacacaa ccaatgcatt tcctggagaa tatatcccta ctgtctttga
      601 caattattct gccaatgtta tggtagatgg aaaaccggtg aatctgggct tatgggatac
      661 agctggacaa gaagattatg acagattacg ccccctatcc tatccgcaaa cagatgtgtt
      721 cttaatttgc ttttcccttg tgagtcctgc atcatttgaa aatgtccgtg caaagtggta
      781 tcctgaggtg cggcaccact gtcccaacac tcccatcatc ctagtgggaa ctaaacttga
      841 tcttagggat gataaagaca cgatcgagaa actgaaggag aagaagctga ctcccatcac
      901 ctatccgcag ggtctagcca tggctaagga gattggtgct gtaaaatacc tggagtgctc
      961 ggcgctcaca cagcgaggcc tcaagacagt gtttgacgaa gcgatccgag cagtcctctg
     1021 acagtaaagg tggatacgga tccgaattcg agctccgtcg acaagcttgc ggccgcactc
     1081 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     1141 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1201 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1261 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1321 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1381 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1441 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1501 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1561 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1621 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1681 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1741 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1801 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1861 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1921 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1981 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     2041 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     2101 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     2161 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     2221 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     2281 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     2341 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     2401 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     2461 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2521 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2581 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2641 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     2701 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2761 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2821 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2881 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2941 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3001 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3061 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3121 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3181 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3241 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3301 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3361 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3421 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3481 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3541 tgcgatattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3601 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3661 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3721 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3781 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3841 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3901 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3961 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4021 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4081 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4141 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4201 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4261 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4321 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4381 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4441 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4501 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagt ggtcctcgcc
     4561 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4621 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4681 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4741 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4801 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4861 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4921 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4981 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     5041 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     5101 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5161 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5221 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5281 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5341 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5401 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5461 taatgatcag tccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5521 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5581 atttaatcgc tgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5641 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5701 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5761 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5821 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5881 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5941 tcctgcatta ggaagcagcc cagtagt
//
