LOCUS       1_CMV_3xFLAG_NLS        5395 bp    DNA     circular SYN 05-JAN-2026
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5395)
  AUTHORS   Weatherall Institute
  TITLE     Direct Submission
  JOURNAL   Exported Jan 5, 2026 from SnapGene 8.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             42..107
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             114..134
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             159..1136
                     /codon_start=1
                     /label=ISCro4 (human codon optimised)
                     /translation="MEQELHFIGIDVSKAKLDVDVLRPDGRHRSKKFANTPKGHDELLR
                     WLSGHRVAPAHICMEATSTYMEDVAAHLSDAGYTVSVINPALGKAFAQSEGLRSKTDAV
                     DARMLAEFCRQKRPPAWEAPHPVERALRALVLRHQSLTDMHTQELNRLETAREVQRPSI
                     DAHLLWLHAELKRIEKQIKDLTDDDPDMKHRRKLLESIPGIGEKTSAVLLAYTGLKERF
                     THARQFAAFAGLTPRRYESGSSVNRASRMSKAGHASLRRALYMPAMVAVSKTEWGRAFR
                     DRLAGNGKKGKVIIGAMMRKLAQVAYGVLKSGVPFDASRHNPVAA"
     CDS             1149..1196
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    1264..1385
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     terminator      1500..1546
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1576..1712
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1757..1828
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2054..2642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             join(3388..3456,3457..4248)
                     /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"
     terminator      4261..4397
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    4549..4620
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(4625..4671)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    4689..4737
                     /note="synthetic polyadenylation signal"
     enhancer        4818..5121
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5123..5325
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 taatacgact cactataggg agagccgcca ccgccaccat ggactataag gaccacgacg
       61 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga
      121 agaagcggaa ggtcggtatc cacggagtcc cagcagccat ggagcaggag ctgcacttca
      181 tcggaatcga cgtgagcaaa gctaagctcg acgtggatgt cttaagaccc gacggaaggc
      241 atcgctcaaa aaagtttgcc aacacaccta aaggacacga tgaactgctg cggtggctga
      301 gcggtcaccg agtagcccct gcacacatct gcatggaggc tacgagtacc tatatggaag
      361 acgtggccgc ccacctgtca gacgccggat acactgtgtc tgtaattaac cccgcgctgg
      421 gcaaggcctt tgcgcagagc gaaggattgc ggtccaagac tgacgcagtg gatgcacgaa
      481 tgcttgccga gttttgtaga cagaagagac caccagcctg ggaagcccca cacccagtag
      541 agcgcgcact gcgagccctg gtcctgcggc accagagctt gactgatatg cacactcagg
      601 agctgaaccg ccttgagacc gctcgggaag tgcagcggcc ctcaatagac gcccatctcc
      661 tgtggctgca cgcggaactg aaaaggatag aaaagcagat aaaggatctg actgatgatg
      721 acccagacat gaaacatagg cgcaaattgc tggaatctat cccaggtatc ggagaaaaga
      781 cctccgctgt cttgctggcc tacactggac tgaaggagcg gttcacacac gcaaggcaat
      841 tcgccgcttt cgctggtctg acaccacgcc ggtatgaaag tgggtcatca gtcaatcggg
      901 cgtctcgcat gagtaaggcc ggccacgcct cccttaggag agctttgtac atgccggcca
      961 tggtagcggt gtctaagact gagtggggcc gcgcgttccg cgaccgactg gcaggaaatg
     1021 gcaaaaaggg taaggtgatt atcggggcta tgatgagaaa gctggcacag gtggcctacg
     1081 gtgtactgaa gagtggcgtg ccatttgacg cttccagaca caatcccgtg gccgcatctc
     1141 gggccgataa gagacccgct gctaccaaaa aagctgggca agcaaagaaa aaaaaagagt
     1201 tcgggagtgg ctaagctagc ttgactgact gagatacagc gtaccttcag ctcacagaca
     1261 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     1321 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     1381 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     1441 ttttttaaag caagtaaaac ctctacaaat gtggtattgg cccatctcta tcggtatcgt
     1501 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgtgcc cctcgggccg
     1561 gattgctatc taccggcatt ggcgcagaaa aaaatgcctg atgcgacgct gcgcgtctta
     1621 tactcccaca tatgccagat tcagcaacgg atacggcttc cccaacttgc ccacttccat
     1681 acgtgtcctc cttaccagaa atttatcctt aaggtcgtca gctatcctgc aggcgatctc
     1741 tcgatttcga tcaagacatt cctttaatgg tcttttctgg acaccactag gggtcagaag
     1801 tagttcatca aactttcttc cctccctaat ctcattggtt accttgggct atcgaaactt
     1861 aattaaccag tcaagtcagc tacttggcga gatcgacttg tctgggtttc gactacgctc
     1921 agaattgcgt cagtcaagtt cgatctggtc cttgctattg cacccgttct ccgattacga
     1981 gtttcattta aatcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2041 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2101 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2161 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2221 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2281 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2341 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2401 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2461 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2521 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2581 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2641 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2701 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2761 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2821 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcat
     2881 ttaaatttcc gaactctcca aggccctcgt cggaaaatct tcaaaccttt cgtccgatcc
     2941 atcttgcagg ctacctctcg aacgaactat cgcaagtctc ttggccggcc ttgcgccttg
     3001 gctattgctt ggcagcgcct atcgccaggt attactccaa tcccgaatat ccgagatcgg
     3061 gatcacccga gagaagttca acctacatcc tcaatcccga tctatccgag atccgaggaa
     3121 tatcgaaatc ggggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg agtctcgacg
     3181 atccatatcg ttgcttggca gtcagccagt cggaatccag cttgggaccc aggaagtcca
     3241 atcgtcagat attgtactca agcctggtca cggcagcgta ccgatctgtt taaacctaga
     3301 tattgatagt ctgatcggtc aacgtataat cgagtcctag cttttgcaaa catctatcaa
     3361 gagacaggat cagcaggagg ctttcgcatg agtattcaac atttccgtgt cgcccttatt
     3421 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3481 aaagatgctg aagatcagtt gggtgcgcga gtgggttaca tcgaactgga tctcaacagc
     3541 ggtaagatcc ttgagagttt tcgccccgaa gaacgctttc caatgatgag cacttttaaa
     3601 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     3661 cgcatacact attctcagaa tgacttggtt gagtattcac cagtcacaga aaagcatctt
     3721 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3781 gcggccaact tacttctgac aacgattgga ggaccgaagg agctaaccgc ttttttgcac
     3841 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3901 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacctt gcgtaaacta
     3961 ttaactggcg aactacttac tctagcttcc cggcaacagt tgatagactg gatggaggcg
     4021 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4081 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4141 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4201 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaacc gattctaggt
     4261 gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc
     4321 cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac
     4381 cagaaattta tccttaagat cccgaatcgt ttaaactcga ctctggctct atcgaatctc
     4441 cgtcgtttcg agcttacgcg aacagccgtg gcgctcattt gctcgtcggg catcgaatct
     4501 cgtcagctat cgtcagctta cctttttggc agcgatcgcg gctcccgaca tcttggacca
     4561 ttagctccac aggtatcttc ttccctctag tggtcataac agcagcttca gctacctctc
     4621 aattcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct atcaatcgtt
     4681 gcgttacaca cacaaaaaac caacacacat ccatcttcga tggatagcga ttttattatc
     4741 taactgctga tcgagtgtag ccagatctag taatcaatta cggggtcatt agttcatagc
     4801 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     4861 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     4921 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     4981 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     5041 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     5101 ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acatcaatgg gcgtggatag
     5161 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     5221 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     5281 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
     5341 cagatcagat ctttgtcgat cctaccatcc actcgacaca cccgccagcg gccgc
//
