LOCUS       sequence-217226-        5564 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and Bla (beta-lactamase from the halophile 
            Chromohalobacter sp. fusions with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5564)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5564)
  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            "unpublished"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-Bla
FEATURES             Location/Qualifiers
     source          1..5564
                     /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             1281..1304
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(1318..1336)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1319..1336)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1394..1412)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1408..1455
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1691..1712)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1862..2944)
                     /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     2906..2925
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2945..3022)
                     /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     3161..3180
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3334..3353)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3453..3475
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3513..3531)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3599..3703
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3704..3772,3773..4564)
                     /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(3922..3941)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4735..5323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5224..5243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5477..5494
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc gacgatgcca gcgaccgcca gacgcgtgaa gtgctggacc cgatcgtcgc
      181 gagcctgatg gaagcccagc aaatcccggg tatggcgatt gcgctggttc gtccagaggg
      241 tactacgatc agccactacg gtgcggcgga tcgtgagact ggtacgccgg ttgacgacga
      301 taccctgttt gagattggca gcctgagcaa aacgctgacc gcgaccctgg cttccctggc
      361 ggaagtcgaa ggcaaactgg acttcgatgc cccggtgagc cgctatctgc cggagctgga
      421 aggtagcgcg ttcgacgata tctccggtct gaatctgggc acccacacgg gtggcggtct
      481 gccgttgttc gttccggacg aggttaccga tcgtgcatct ctgatggcat ggtaccgcga
      541 atggcaaccg accgagccaa tcggtgagag ccgcacctac agcaatctgg gtattggttt
      601 gctgggtttg gaaacggcag cttcgctgga cggcgagttt gtgccgacca tgcgtgccaa
      661 ggtgctggca ccgctgggta tgcaagatac ctggtacgat gtcccagaag cccgcatggc
      721 cgactacgcg atgggtgagg ataaagacgg ccagccgacg cgtgtgagcc cgggtgttct
      781 ggatgacgag gcatacggca ttaagaccac cgctgcggac ctggcgaaac tggtccgtgc
      841 caatctgcat ctggcggatg ttgatgcgga gctgcagcag gcaatcgatg cgacccgcca
      901 gggccattac cgtgtcggtg acatgacgca agctctgatc tgggaacaat atagcctgcc
      961 tgttgcaccg gaaaccctgc gtgcgggcaa cggctacgac atgattttgg agccgaatgc
     1021 ggcagaggcg ctggaaccgc ctcaatcccc gcgtgatgac gtgtgggtca acaaaacggg
     1081 cagcaccaac ggttttggtg gttatatcgt catgctgccg ggtaagcaca ccggcctggt
     1141 gatgctggca aacaagaatt atccgaacga cgcgcgtgtt gaggccgcat atcgcattct
     1201 gagcggcctg ggcgcgattg atgttccgag cggtaccgaa aacctgtact tccagtgaga
     1261 ccttaattaa ctcgagcgca tggagccacc cgcagttcga aaaataagct tgagtattct
     1321 atagtgtcac ctaaatccca gcttgatccg gctgctaaca aagcccgaaa ggaagctgag
     1381 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1441 ttgaggggtt ttttgctgaa aggaggaact atatccggat aacctggcgt aatagcgaag
     1501 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt
     1561 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     1621 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     1681 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     1741 agggcgaaaa accgtctatc agggcgatgg cccacagctt gcatgcctgc aggtcggaag
     1801 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     1861 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1921 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     1981 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     2041 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     2101 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     2161 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     2221 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     2281 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     2341 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     2401 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     2461 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     2521 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     2581 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     2641 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     2701 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     2761 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     2821 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     2881 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     2941 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     3001 ttttgcacca ttcgatggtg tcggcgagct cgaattgtga accatcaccc taatcaagtt
     3061 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3121 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3181 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3241 cgcttaatgc gccgctacag ggcgcgtcct gatgcggtat tttctcctta cgcatctgtg
     3301 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3361 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3421 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3481 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     3541 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     3601 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3661 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     3721 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     3781 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3841 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3901 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3961 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4021 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4081 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4141 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4201 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4261 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4321 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     4381 ctggtttatt gctgataaat ctggagccgg tgagcgtggt tctcgcggta tcattgcagc
     4441 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     4501 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     4561 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     4621 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4681 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4741 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4801 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4861 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4921 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4981 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5041 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5101 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5161 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5221 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5281 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5341 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5401 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     5461 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     5521 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tgat
//
