LOCUS       sequence-247820-        4892 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 4892)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4892)
  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            "unpublished"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-Spy
FEATURES             Location/Qualifiers
     source          1..4892
                     /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..115
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             609..632
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(646..664)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(647..664)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(722..740)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      736..783
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1019..1040)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1190..2272)
                     /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     2234..2253
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2273..2350)
                     /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     2489..2508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2662..2681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2781..2803
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2841..2859)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2927..3031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3032..3100,3101..3892)
                     /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(3250..3269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4063..4651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4552..4571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4805..4822
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggt gcagacacca ctaccgcagc accggctgac gcgaagccga tgatgcacca
      181 caaaggcaag ttcggtccgc atcaggacat gatgttcaaa gacctgaacc tgaccgacgc
      241 gcagaaacag cagatccgcg aaatcatgaa aggccagcgt gaccagatga aacgtccgcc
      301 gctggaagaa cgccgcgcaa tgcatgacat cattgccagc gataccttcg ataaagtaaa
      361 agctgaagcg cagatcgcaa aaatggaaga acagcgcaaa gctaacatgc tggcgcacat
      421 ggaaacccag aacaaaattt acaacatcct gacgccggaa cagaaaaagc aatttaatgc
      481 taattttgag aagcgtctga cagaacgtcc agcggcaaaa ggtaaaatgc ctgcaactgc
      541 tgaaagtggt acctccggtt ctactgaaaa cctgtacttc cagtgagacc ttaattaact
      601 cgagcgcatg gagccacccg cagttcgaaa aataagcttg agtattctat agtgtcacct
      661 aaatcccagc ttgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      721 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      781 ttgctgaaag gaggaactat atccggataa cctggcgtaa tagcgaagag gcccgcaccg
      841 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat
      901 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
      961 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     1021 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1081 cgtctatcag ggcgatggcc cacagcttgc atgcctgcag gtcggaagca taaagtgtaa
     1141 agcctggggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     1201 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1261 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     1321 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     1381 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     1441 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     1501 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     1561 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1621 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1681 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1741 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1801 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1861 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1921 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1981 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     2041 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     2101 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     2161 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     2221 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     2281 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     2341 cgatggtgtc ggcgagctcg aattgtgaac catcacccta atcaagtttt ttggggtcga
     2401 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2461 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2521 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2581 cgctacaggg cgcgtcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2641 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     2701 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2761 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2821 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2881 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2941 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3001 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3061 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3121 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3181 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3241 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3301 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3361 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3421 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3481 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3541 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3601 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3661 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3721 tgataaatct ggagccggtg agcgtggttc tcgcggtatc attgcagcac tggggccaga
     3781 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3841 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3901 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3961 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4021 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4081 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4141 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4201 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4261 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4321 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4381 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4441 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4501 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4561 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4621 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4681 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4741 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4801 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     4861 cgcgcgttgg ccgattcatt aatgcagctg at
//
