LOCUS       sequence-467624-        5026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Strep and His-tagged Evasin 4 (EVA4_RHISA) in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5026)
  AUTHORS   Kryukova J, Vales S, Payne M, Smagurauskaite G, Chandra S, Clark CJ,
            Davies G, Bhattacharya S
  TITLE     Development of chemokine network inhibitors using combinatorial 
            saturation mutagenesis.
  JOURNAL   Commun Biol. 2025 Apr 3;8(1):549. doi: 10.1038/s42003-025-07778-6.
  PUBMED    40181178
REFERENCE   2  (bases 1 to 5026)
  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: "Commun 
            Biol."; date: "2025-04-3"; volume: "8(1):549. doi"; pages: " 
            10.1038/s42003-025-07778-6"
COMMENT     Sequence Label: P1909:EVA4_RHISA-StrepII-8xHis
FEATURES             Location/Qualifiers
     source          1..5026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             52..75
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             82..105
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(211..230)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    276..331
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(330..349)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(692..708)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(692..708)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(705..727)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    716..732
                     /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)."
     promoter        complement(join(740..746,747..764,765..770))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    785..806
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(935..952)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1106..1694)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1186..1205)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1865..2656,2657..2725))
                     /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     2488..2507
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2726..2830)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        2861..3240
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3242..3517
                     /label=chicken beta-actin promoter
     intron          3518..4534
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4458..4480
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4542..4561
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      4616..4625
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 accggtggcg gaggaggctc tggtggtgga ggttctggag gcgcaagcgc gtggagccac
       61 ccgcagttcg aaaaactcga gcaccaccat caccatcacc atcactaact cgagactagt
      121 atcgcgataa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
      181 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
      241 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
      301 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
      361 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
      421 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
      481 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
      541 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
      601 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct
      661 cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
      721 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
      781 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
      841 gaaacctgtc gtgccagcgg atcgatccgc tgcattaatg aatcggccaa cgcgcgggga
      901 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      961 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1021 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1081 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1141 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1201 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1261 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1321 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1381 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1441 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1501 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1561 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1621 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1681 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1741 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1801 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1861 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1921 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1981 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     2041 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2101 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2161 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2221 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2281 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2341 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2401 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2461 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2521 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2581 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2641 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2701 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2761 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2821 gggttccgcg cacatttccc cgaaaagtgc cacctgggtc gacattgatt attgactagt
     2881 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     2941 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     3001 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     3061 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     3121 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     3181 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     3241 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
     3301 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg
     3361 gggcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
     3421 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
     3481 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg
     3541 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
     3601 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt
     3661 ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg
     3721 ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc
     3781 cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg
     3841 tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg
     3901 gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg
     3961 tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca
     4021 cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg
     4081 cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg
     4141 ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg
     4201 cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat
     4261 ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa
     4321 gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg
     4381 ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg
     4441 gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc
     4501 taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt
     4561 gctgtctcat cattttggca aagaattgcg gccgtctcag gccgaattca agcttgccac
     4621 catggggatc cttcccagcc ctgggatgcc tgcgctgctc tccctcgtga gccttctctc
     4681 cgtgctgctg atgggttgcg tagctgaaac cggtgaagtg ccacaaatga ctagctcctc
     4741 cgcgccagat cttgaagagg aggacgatta cacagcatat gcaccgctga cgtgctattt
     4801 tacaaattcc acgcttggtc tgctggcccc cccaaattgc tccgtgcttt gcaacagtac
     4861 cacaacttgg tttaatgaaa cttcgccaaa caatgcttcg tgtttgctga ctgtggattt
     4921 tcttacacag gacgccattc tacaagaaaa ccaaccgtac aactgcagtg tgggacactg
     4981 tgataatggg acttgcgccg ggccccctcg acacgctcag tgctgg
//
