LOCUS       sequence-247825-        4499 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 4499)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4499)
  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            "unpublished"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-Stag
FEATURES             Location/Qualifiers
     source          1..4499
                     /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             89..133
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             140..163
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             216..239
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(253..271)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(254..271)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(329..347)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      343..390
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(626..647)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(797..1879)
                     /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     1841..1860
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1880..1957)
                     /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     2096..2115
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2269..2288)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2388..2410
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2448..2466)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2534..2638
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2639..2707,2708..3499)
                     /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(2857..2876)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3670..4258
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4159..4178
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4412..4429
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa agaaaccgct gctgctaaat tcgaacgcca
      121 gcacatggac agcactagtc accatcacca tcaccatcac cattctggta ccgaaaacct
      181 gtacttccag tgagacctta attaactcga gcgcatggag ccacccgcag ttcgaaaaat
      241 aagcttgagt attctatagt gtcacctaaa tcccagcttg atccggctgc taacaaagcc
      301 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
      361 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggataacct
      421 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
      481 gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
      541 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
      601 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
      661 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac agcttgcatg
      721 cctgcaggtc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaacttac
      781 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
      841 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
      901 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
      961 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     1021 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     1081 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     1141 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     1201 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     1261 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     1321 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     1381 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     1441 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     1501 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     1561 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     1621 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     1681 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     1741 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     1801 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     1861 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     1921 ccataccgcg aaaggttttg caccattcga tggtgtcggc gagctcgaat tgtgaaccat
     1981 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2041 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2101 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2161 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcctgatgc ggtattttct
     2221 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     2281 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     2341 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2401 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     2461 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     2521 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2581 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2641 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2701 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2761 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2821 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2881 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2941 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3001 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3061 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3121 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3181 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3241 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3301 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggttctcg
     3361 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3421 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3481 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3541 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3601 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3661 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3721 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3781 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     3841 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3901 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3961 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4021 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4081 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4141 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4201 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4261 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4321 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4381 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4441 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctgat
//
