LOCUS       sequence-220209-        4766 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Used for expression of protein in E. coli as N-terminal Sumo tag 
            fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4766)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4766)
  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: pOP3SU
FEATURES             Location/Qualifiers
     source          1..4766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..76
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        57..75
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    75..99
                     /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             112..134
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             148..192
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             202..225
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             232..525
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     promoter        complement(572..590)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(573..590)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(648..666)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      662..709
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(945..966)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1116..2198)
                     /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     2160..2179
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2199..2276)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     2419..2438
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2592..2611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2711..2733
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2771..2789)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2857..2961
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2962..3030,3031..3822)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3180..3199)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3993..4581
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4482..4501
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4735..4752
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctgattaat
       61 acgactcact atagggaatt gtgagcggat aacaattcct ctagaaataa ttttgtttaa
      121 ctttaagaag gagatatatc catgaatgga ctgaatgata tctttgaagc gcagaaaatt
      181 gaatggcatg aatccggatc tcatcaccat caccatcacc atcacactag tatgtcggac
      241 tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc tgagactcac
      301 atcaatttaa aggtgtccga tggatcttca gagatcttct tcaagatcaa aaagaccact
      361 cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat ggactcctta
      421 agattcttgt acgacggtat tagaattcaa gctgatcaga cccctgaaga tttggacatg
      481 gaggataacg atattattga ggctcacaga gaacagattg gtggatccat ggaattcgcg
      541 gccgccctag gctcgagcta agcttgagta ttctatagtg tcacctaaat cccagcttga
      601 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
      661 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
      721 aactatatcc ggataacctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
      781 cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg
      841 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
      901 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
      961 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     1021 atggcccaca gcttgcatgc ctgcaggtcg gaagcataaa gtgtaaagcc tggggtgcct
     1081 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     1141 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1201 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     1261 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     1321 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     1381 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     1441 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     1501 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     1561 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     1621 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     1681 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     1741 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     1801 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     1861 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1921 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1981 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2041 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     2101 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     2161 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     2221 tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat ggtgtcgggt
     2281 accgagctcg aattgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     2341 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     2401 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     2461 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     2521 cgcgtcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
     2581 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     2641 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     2701 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     2761 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     2821 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2881 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2941 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3001 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3061 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3121 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3181 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3241 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3301 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3361 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3421 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3481 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3541 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3601 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3661 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3721 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3781 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3841 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3901 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3961 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4021 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4081 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4141 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4201 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4261 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4321 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4381 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4441 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4501 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4561 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4621 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4681 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4741 tcagtgagcg aggaagcgga agagcg
//
