LOCUS       sequence-252331-        4898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4898)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4898)
  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"; journalName: 
            "unpublished"
COMMENT     Sequence Label: pExp-Skp
FEATURES             Location/Qualifiers
     source          1..4898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..23
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4..22
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    22..46
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             95..118
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             618..641
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(655..673)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(656..673)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(731..749)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      745..792
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1028..1049)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1199..2281)
                     /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     2243..2262
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2282..2359)
                     /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     2498..2517
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2671..2690)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2790..2812
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2850..2868)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2936..3040
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3041..3109,3110..3901)
                     /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(3259..3278)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4072..4660
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4561..4580
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4814..4831
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gattaatacg actcactata gggaattgtg agcggataac aattcctcta gaaataattt
       61 tgtttaactt taagaaggag atatatccat gaatcaccat caccatcacc atcaccattc
      121 tggcactagt ggtgctgaca aaattgcaat cgtcaacatg ggcagcctgt tccagcaggt
      181 agcgcagaaa accggtgttt ctaacacgct ggaaaatgag ttcaaaggcc gtgccagcga
      241 actgcagcgt atggaaaccg atctgcaggc taaaatgaaa aagctgcagt ccatgaaagc
      301 gggcagcgat cgcactaagc tggaaaaaga cgtgatggct cagcgccaga cttttgctca
      361 gaaagcgcag gcttttgagc aggatcgcgc acgtcgttcc aacgaagaac gcggcaaact
      421 ggttactcgt atccagactg ctgtgaaatc cgttgccaac agccaggata tcgatctggt
      481 tgttgatgca aacgccgttg cttacaacag cagcgatgta aaagacatca ctgccgacgt
      541 actgaaacag gttaaaggta cctccggttc tactgaaaac ctgtacttcc agtgagacct
      601 taattaactc gagcgcatgg agccacccgc agttcgaaaa ataagcttga gtattctata
      661 gtgtcaccta aatcccagct tgatccggct gctaacaaag cccgaaagga agctgagttg
      721 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      781 aggggttttt tgctgaaagg aggaactata tccggataac ctggcgtaat agcgaagagg
      841 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag
      901 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
      961 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     1021 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     1081 gcgaaaaacc gtctatcagg gcgatggccc acagcttgca tgcctgcagg tcggaagcat
     1141 aaagtgtaaa gcctggggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     1201 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1261 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     1321 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     1381 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     1441 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     1501 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     1561 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     1621 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     1681 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     1741 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     1801 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     1861 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     1921 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     1981 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     2041 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     2101 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     2161 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     2221 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     2281 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     2341 tgcaccattc gatggtgtcg gcgagctcga attgtgaacc atcaccctaa tcaagttttt
     2401 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     2461 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     2521 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     2581 ttaatgcgcc gctacagggc gcgtcctgat gcggtatttt ctccttacgc atctgtgcgg
     2641 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2701 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2761 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2821 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     2881 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     2941 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3001 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3061 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3121 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3181 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3241 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3301 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3361 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3421 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3481 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3541 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3601 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3661 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3721 gtttattgct gataaatctg gagccggtga gcgtggttct cgcggtatca ttgcagcact
     3781 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3841 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3901 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3961 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4021 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4081 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4141 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4201 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4261 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4321 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4381 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4441 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4501 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4561 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4621 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4681 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4741 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4801 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     4861 gcctctcccc gcgcgttggc cgattcatta atgcagct
//
