LOCUS       sequence-252355-        4472 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 4472)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4472)
  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            "unpublished"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-Etag
FEATURES             Location/Qualifiers
     source          1..4472
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..38
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        19..37
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    37..61
                     /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             74..96
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             131..154
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             207..230
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(244..262)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(245..262)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(320..338)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      334..381
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(617..638)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(788..1870)
                     /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     1832..1851
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1871..1948)
                     /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     2087..2106
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2260..2279)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2379..2401
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2439..2457)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2525..2629
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2630..2698,2699..3490)
                     /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(2848..2867)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3661..4249
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4150..4169
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4403..4420
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttcattaatg cagctgatta atacgactca ctatagggaa ttgtgagcgg ataacaattc
       61 ctctagaaat aattttgttt aactttaaga aggagatata tccatgacag atgtaacgat
      121 taaaactagt caccatcacc atcaccatca ccattctggt accgaaaacc tgtacttcca
      181 gtgagacctt aattaactcg agcgcatgga gccacccgca gttcgaaaaa taagcttgag
      241 tattctatag tgtcacctaa atcccagctt gatccggctg ctaacaaagc ccgaaaggaa
      301 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      361 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggataacc tggcgtaata
      421 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
      481 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
      541 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
      601 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
      661 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca cagcttgcat gcctgcaggt
      721 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactta cattaattgc
      781 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
      841 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
      901 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
      961 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     1021 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     1081 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     1141 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     1201 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     1261 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     1321 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     1381 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     1441 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     1501 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     1561 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     1621 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     1681 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     1741 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     1801 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     1861 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     1921 gaaaggtttt gcaccattcg atggtgtcgg cgagctcgaa ttgtgaacca tcaccctaat
     1981 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2041 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2101 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2161 ccgccgcgct taatgcgccg ctacagggcg cgtcctgatg cggtattttc tccttacgca
     2221 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     2281 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2341 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2401 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     2461 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2521 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2581 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2641 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2701 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2761 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2821 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2881 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2941 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3001 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3061 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3121 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3181 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3241 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3301 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttctc gcggtatcat
     3361 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3421 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3481 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3541 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3601 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3661 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3721 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3781 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     3841 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3901 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3961 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4021 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4081 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4141 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4201 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4261 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4321 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4381 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     4441 acgcaaaccg cctctccccg cgcgttggcc ga
//
