LOCUS       sequence-220666-        5948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and NusA fusions with optional C-terminal 
            StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5948)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5948)
  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            "unpublished"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-NusA
FEATURES             Location/Qualifiers
     source          1..5948
                     /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             131..1612
                     /codon_start=1
                     /gene="nusA"
                     /product="transcription elongation protein (N utilization 
                     substance protein A)"
                     /label=NusA
                     /note="highly soluble in E. coli"
                     /translation="NKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVR
                     VQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRI
                     TTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILR
                     EDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEI
                     KAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQ
                     FVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTV
                     DDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDE
                     PTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDL
                     AEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     CDS             1665..1688
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(1702..1720)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1703..1720)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1778..1796)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1792..1839
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2075..2096)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2246..3328)
                     /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     3290..3309
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3329..3406)
                     /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     3545..3564
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3718..3737)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3837..3859
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3897..3915)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3983..4087
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4088..4156,4157..4948)
                     /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(4306..4325)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5119..5707
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5608..5627
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5861..5878
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc aacaaagaaa ttttggctgt agttgaagcc gtatccaatg aaaaggcgct
      181 acctcgcgag aagattttcg aagcattgga aagcgcgctg gcgacagcaa caaagaaaaa
      241 atatgaacaa gagatcgacg tccgcgtaca gatcgatcgc aaaagcggtg attttgacac
      301 tttccgtcgc tggttagttg ttgatgaagt cacccagccg accaaggaaa tcacccttga
      361 agccgcacgt tatgaagatg aaagcctgaa cctgggcgat tacgttgaag atcagattga
      421 gtctgttacc tttgaccgta tcactaccca gacggcaaaa caggttatcg tgcagaaagt
      481 gcgtgaagcc gaacgtgcga tggtggttga tcagttccgt gaacacgaag gtgaaatcat
      541 caccggcgtg gtgaaaaaag taaaccgcga caacatctct ctggatctgg gcaacaacgc
      601 tgaagccgtg atcctgcgcg aagatatgct gccgcgtgaa aacttccgcc ctggcgaccg
      661 cgttcgtggc gtgctctatt ccgttcgccc ggaagcgcgt ggcgcgcaac tgttcgtcac
      721 tcgttccaag ccggaaatgc tgatcgaact gttccgtatt gaagtgccag aaatcggcga
      781 agaagtgatt gaaattaaag cagcggctcg cgatccgggt tctcgtgcga aaatcgcggt
      841 gaaaaccaac gataaacgta tcgatccggt aggtgcttgc gtaggtatgc gtggcgcgcg
      901 tgttcaggcg gtgtctactg aactgggtgg cgagcgtatc gatatcgtcc tgtgggatga
      961 taacccggcg cagttcgtga ttaacgcaat ggcaccggca gacgttgctt ctatcgtggt
     1021 ggatgaagat aaacacacta tggacatcgc cgttgaagcc ggtaatctgg cgcaggcgat
     1081 tggccgtaac ggtcagaacg tgcgtctggc ttcgcaactg agcggttggg aactcaacgt
     1141 gatgaccgtt gacgacctgc aagctaagca tcaggcggaa gcgcacgcag cgatcgacac
     1201 cttcaccaaa tatctcgaca tcgacgaaga cttcgcgact gttctggtag aagaaggctt
     1261 ctcgacgctg gaagaattgg cctatgtgcc gatgaaagag ctgttggaaa tcgaaggcct
     1321 tgatgagccg accgttgaag cactgcgcga gcgtgctaaa aatgcactgg ccaccattgc
     1381 acaggcccag gaagaaagcc tcggtgataa caaaccggct gacgatctgc tgaaccttga
     1441 aggggtagat cgtgatttgg cattcaaact ggccgcccgt ggcgtttgta cgctggaaga
     1501 tctcgccgaa cagggcattg atgatctggc tgatatcgaa gggttgaccg acgaaaaagc
     1561 cggagcactg attatggctg cccgtaatat ttgctggttc ggtgacgaag cgagcggtac
     1621 cgaaaacctg tacttccagt gagaccttaa ttaactcgag cgcatggagc cacccgcagt
     1681 tcgaaaaata agcttgagta ttctatagtg tcacctaaat cccagcttga tccggctgct
     1741 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1801 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1861 ggataacctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     1921 gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     1981 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     2041 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     2101 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccaca
     2161 gcttgcatgc ctgcaggtcg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2221 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2281 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     2341 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     2401 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     2461 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     2521 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     2581 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     2641 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     2701 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     2761 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     2821 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     2881 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     2941 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     3001 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     3061 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     3121 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     3181 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     3241 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     3301 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     3361 gctatcatgc cataccgcga aaggttttgc accattcgat ggtgtcggcg agctcgaatt
     3421 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     3481 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     3541 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     3601 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcctgatgcg
     3661 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
     3721 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     3781 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3841 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     3901 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     3961 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4021 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4081 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4141 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4201 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4261 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4321 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4381 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4441 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4501 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4561 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4621 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4681 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4741 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4801 tggttctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4861 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4921 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4981 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5041 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5101 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5161 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5221 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5281 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5341 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5401 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5461 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5521 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5581 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5641 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5701 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5761 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5821 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5881 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5941 cagctgat
//
