LOCUS       sequence-217216-        4760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and AFV (Acidianus Filamentous Virus 1 protein) 
            fusions with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4760)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4760)
  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            "unpublished"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-AFV
FEATURES             Location/Qualifiers
     source          1..4760
                     /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             477..500
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(514..532)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(515..532)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(590..608)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      604..651
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(887..908)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1058..2140)
                     /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     2102..2121
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2141..2218)
                     /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     2357..2376
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2530..2549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2649..2671
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2709..2727)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2795..2899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2900..2968,2969..3760)
                     /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(3118..3137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3931..4519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4420..4439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4673..4690
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc gacacccacg aatttcataa gctgctgatc aaagttgtcg acctgttctt
      181 ggaagatcgt atcaaagagt ttgagctgaa attgaacacg accctggatg agctggaatt
      241 tgaagaactg atcggcaaac cggacagcag caacagcgcc gaaaacaatg gtattttcat
      301 cgacgagtat tcttacgatg cgtccgagaa cgcaattaag aagctgttcg tcgagtacgt
      361 tcgccaaccg gagtttaagt ataccgttct gagcattaaa ggcgtgaatg attgggtgcg
      421 tgaaagcggt accgaaaacc tgtacttcca gtgagacctt aattaactcg agcgcatgga
      481 gccacccgca gttcgaaaaa taagcttgag tattctatag tgtcacctaa atcccagctt
      541 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
      601 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
      661 ggaactatat ccggataacc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
      721 aacagttgcg cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt
      781 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
      841 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
      901 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
      961 cgatggccca cagcttgcat gcctgcaggt cggaagcata aagtgtaaag cctggggtgc
     1021 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1081 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1141 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     1201 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     1261 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     1321 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     1381 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     1441 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     1501 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     1561 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     1621 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     1681 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     1741 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     1801 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     1861 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     1921 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     1981 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     2041 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     2101 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     2161 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcaccattcg atggtgtcgg
     2221 cgagctcgaa ttgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     2281 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2341 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2401 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2461 cgtcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     2521 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     2581 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     2641 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     2701 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2761 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2821 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2881 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2941 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3001 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3061 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3121 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3181 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3241 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3301 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3361 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3421 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3481 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3541 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3601 agccggtgag cgtggttctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3661 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3721 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3781 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3841 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3901 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3961 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4021 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4081 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4141 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4201 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4261 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4321 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4381 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4441 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4501 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4561 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4621 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4681 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     4741 gattcattaa tgcagctgat
//
