LOCUS       sequence-252326-        4910 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 4910)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4910)
  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            "unpublished"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-Calm
FEATURES             Location/Qualifiers
     source          1..4910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    7..31
                     /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             44..66
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             80..103
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             116..559
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTE
                     AELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             615..638
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(652..670)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(653..670)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(728..746)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      742..789
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1025..1046)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1196..2278)
                     /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     2240..2259
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2279..2356)
                     /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     2495..2514
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2668..2687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2787..2809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2847..2865)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2933..3037
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3038..3106,3107..3898)
                     /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(3256..3275)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4069..4657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4558..4577
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4811..4828
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(4899..4910,1..8)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4899..4910,1..7)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctatagggaa ttgtgagcgg ataacaattc ctctagaaat aattttgttt aactttaaga
       61 aggagatata tccatgaatc accatcacca tcaccatcac cattctggca ctagtgctga
      121 ccagctcacc gaagaacaga tcgcggagtt caaagaggcg ttctctctgt tcgacaagga
      181 cggcgacggc accatcacca ccaaagaact gggtacggtg atgcgctctc tgggtcagaa
      241 cccgaccgag gcggaactgc aggacatgat caacgaagta gacgcggacg gtaacggtac
      301 gatcgacttc ccggaatttc tcaccatgat ggcgcgtaaa atgaaagaca ccgactctga
      361 agaagaaatc cgtgaagcgt tccgtgtttt cgataaagac ggcaacggtt acatctctgc
      421 ggcggagctg cgtcacgtta tgaccaacct gggtgaaaaa ctgaccgacg aggaagttga
      481 cgaaatgatc cgcgaagccg acatcgacgg tgacggccag gttaactacg aagaatttgt
      541 tcaaatgatg accgcgaaag gctctggtac cgaaaacctg tacttccagt gagaccttaa
      601 ttaactcgag cgcatggagc cacccgcagt tcgaaaaata agcttgagta ttctatagtg
      661 tcacctaaat cccagcttga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      721 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      781 ggttttttgc tgaaaggagg aactatatcc ggataacctg gcgtaatagc gaagaggccc
      841 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt
      901 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
      961 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     1021 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     1081 aaaaaccgtc tatcagggcg atggcccaca gcttgcatgc ctgcaggtcg gaagcataaa
     1141 gtgtaaagcc tggggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     1201 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     1261 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     1321 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     1381 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     1441 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     1501 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     1561 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     1621 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     1681 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     1741 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     1801 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     1861 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     1921 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     1981 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     2041 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     2101 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     2161 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     2221 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     2281 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     2341 accattcgat ggtgtcggcg agctcgaatt gtgaaccatc accctaatca agttttttgg
     2401 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     2461 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     2521 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     2581 atgcgccgct acagggcgcg tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2641 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2701 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2761 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2821 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2881 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2941 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3001 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3061 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3121 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3181 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3241 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3301 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3361 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3421 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3481 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3541 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3601 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3661 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3721 tattgctgat aaatctggag ccggtgagcg tggttctcgc ggtatcattg cagcactggg
     3781 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3841 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3901 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3961 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4021 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4201 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4441 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4561 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4621 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4681 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4741 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4801 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     4861 tctccccgcg cgttggccga ttcattaatg cagctgatta atacgactca
//
