LOCUS       sequence-212066-        4690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminal Avi-His8-tag and GB1 fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4690)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4690)
  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: pOP3BT
FEATURES             Location/Qualifiers
     source          1..4690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..136
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             146..169
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             179..340
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             368..388
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(435..453)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(436..453)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(511..529)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      525..572
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(808..829)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(979..2061)
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2023..2042
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2062..2139)
                     /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     2285..2304
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2460..2479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2579..2601
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2639..2657)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2725..2829
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2830..2898,2899..3690)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCHTLLSRIDAGQEQLGRRARYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3861..4449
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4350..4369
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4603..4620
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tggactgaat gatatctttg aagcgcagaa
      121 aattgaatgg catgaatccg gatctcatca ccatcaccat caccatcaca ctagtaccta
      181 caaactgatc ctgaacggta aaaccctgaa aggtgaaacc accaccgaag ctgtagacgc
      241 tgctactgct gaaaaagttt tcaaacagta cgctaacgac aacggtgtgg acggtgaatg
      301 gacctacgac gacgctacca aaaccttcac ggttacggaa accggtagtg gcaccagtgg
      361 gtcgacagaa aacctgtact tccagggatc catggaattc gcggccgccc taggctcgag
      421 ctaagcttga gtattctata gtgtcaccta aatcccagct tgatccggct gctaacaaag
      481 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
      541 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggataac
      601 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
      661 tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      721 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      781 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      841 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc acagcttgca
      901 tgcctgcagg tcggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactt
      961 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1021 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     1081 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     1141 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     1201 ggttgacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     1261 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     1321 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     1381 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     1441 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     1501 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     1561 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     1621 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     1681 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     1741 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     1801 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     1861 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     1921 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     1981 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     2041 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     2101 tgccataccg cgaaaggttt tgcaccattc gatggtgtcg ggtaccgagc tcgaatttac
     2161 gtgaaccatc acccaaatca agttttttgc ggtcgaggtg ccgtaaagct ctaaatcgga
     2221 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2281 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2341 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcgcctgatg
     2401 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     2461 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     2521 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     2581 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     2641 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2701 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2761 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2821 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2881 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2941 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3001 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtcataca
     3061 ctattatccc gtattgacgc cgggcaagag caactcggtc gccgggcgcg gtattctcag
     3121 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3181 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3241 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3301 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3361 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3421 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3481 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3541 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3601 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3661 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3721 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3781 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3841 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3901 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3961 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4021 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4081 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4141 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4201 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4261 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4321 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4381 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4441 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4501 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4561 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4621 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4681 tgcagctgat
//
