LOCUS       sequence-247817-        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  JOURNAL   unpublished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            "unpublished"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"
     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             128..571
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTE
                     AELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             627..650
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(664..682)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(665..682)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(740..758)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      754..801
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1037..1058)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1208..2290)
                     /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     2252..2271
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2291..2368)
                     /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     2507..2526
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2680..2699)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2799..2821
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2859..2877)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2945..3049
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3050..3118,3119..3910)
                     /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(3268..3287)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4081..4669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4570..4589
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4823..4840
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtgct gaccagctca ccgaagaaca gatcgcggag ttcaaagagg cgttctctct
      181 gttcgacaag gacggcgacg gcaccatcac caccaaagaa ctgggtacgg tgatgcgctc
      241 tctgggtcag aacccgaccg aggcggaact gcaggacatg atcaacgaag tagacgcgga
      301 cggtaacggt acgatcgact tcccggaatt tctcaccatg atggcgcgta aaatgaaaga
      361 caccgactct gaagaagaaa tccgtgaagc gttccgtgtt ttcgataaag acggcaacgg
      421 ttacatctct gcggcggagc tgcgtcacgt tatgaccaac ctgggtgaaa aactgaccga
      481 cgaggaagtt gacgaaatga tccgcgaagc cgacatcgac ggtgacggcc aggttaacta
      541 cgaagaattt gttcaaatga tgaccgcgaa aggctctggt accgaaaacc tgtacttcca
      601 gtgagacctt aattaactcg agcgcatgga gccacccgca gttcgaaaaa taagcttgag
      661 tattctatag tgtcacctaa atcccagctt gatccggctg ctaacaaagc ccgaaaggaa
      721 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      781 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggataacc tggcgtaata
      841 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
      901 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
      961 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     1021 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     1081 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca cagcttgcat gcctgcaggt
     1141 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactta cattaattgc
     1201 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1261 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     1321 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     1381 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     1441 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     1501 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     1561 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     1621 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     1681 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     1741 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     1801 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     1861 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     1921 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     1981 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     2041 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     2101 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     2161 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     2221 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     2281 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     2341 gaaaggtttt gcaccattcg atggtgtcgg cgagctcgaa ttgtgaacca tcaccctaat
     2401 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2461 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2521 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2581 ccgccgcgct taatgcgccg ctacagggcg cgtcctgatg cggtattttc tccttacgca
     2641 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     2701 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2761 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2821 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     2881 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2941 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3001 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3061 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3121 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3181 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3241 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     3301 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3361 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3421 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3481 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3541 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3601 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3661 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3721 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttctc gcggtatcat
     3781 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3841 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3901 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3961 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4021 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4081 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4141 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4201 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4261 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4321 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4381 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4441 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4501 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4561 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4621 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4681 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4741 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4801 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     4861 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgat
//
