LOCUS       sequence-244697-        6278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Artificial phase 2 exon to include a spliced T2A-GeneSwitch effector
            into a locus of interest through RMCE of MiMIC insertion in an 
            intron between coding exons of a gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6278)
  AUTHORS   Gnerer JP, Venken KJ, Dierick HA
  TITLE     Gene-specific cell labeling using MiMIC transposons.
  JOURNAL   Nucleic Acids Res. 2015 Apr 30;43(8):e56. doi: 10.1093/nar/gkv113. 
            Epub 2015 Feb 20.
  PUBMED    25712101
REFERENCE   2  (bases 1 to 6278)
  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: "Nucleic 
            Acids Res."; date: "2015-04-30"; volume: "43(8):e56. doi"; pages: " 
            10.1093/nar/gkv113. Epub 2015 Feb 20"
COMMENT     Sequence Label: pBS_KS_attB2_SA(2)_T2AGeneSwitch
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(83..100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(254..842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(334..353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1013..1804,1805..1873))
                     /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     1636..1655
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1874..1978)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2004..2459)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2141..2162)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2353..2372
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2586..2608
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2600..2617
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2601..2617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2627..2646
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2627..2645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2681..2750
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     CDS             2918..2971
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4547..4918
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     primer_bind     complement(5773..5789)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5773..5789)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    5982..6051
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     complement(6091..6111)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6091..6109)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6130..6146)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6130..6146)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6143..6165)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6154..6170
                     /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(6178..6184,6185..6202,6203..6208))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6223..6244
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
       61 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      121 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      181 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      241 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      301 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      361 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      421 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      481 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      541 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      601 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      661 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      721 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      781 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      841 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      901 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      961 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     1021 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     1081 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     1141 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     1201 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     1261 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     1321 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     1381 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     1441 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     1501 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     1561 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1621 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1681 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1741 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1801 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     1861 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     1921 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     1981 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     2041 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     2101 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     2161 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2221 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2281 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2341 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     2401 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2461 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     2521 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     2581 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     2641 atagggcgaa ttggagctcg ctagcgatgt aggtcacggt ctcgaagccg cggtgcgggt
     2701 gccagggcgt gcccttgggc tccccgggcg cgtactccac ctcacccatc tggtccatca
     2761 tgatggtcga ctctagaagt cgatccaaca tggcgacttg tcccatcccc ggcatgttta
     2821 aatatactaa ttattcttga actaatttta atcaaccgat ttatctctct tccgcaggtc
     2881 gggaggttcc ggtggaagcg gaggtagcgg cggatccgaa ggacgcggaa gcctgttgac
     2941 gtgcggagat gtggaagaga atccaggacc gatgaagcta ctgtcttcta tcgaacaagc
     3001 atgcgatatt tgccgactta aaaagctcaa gtgctccaaa gaaaaaccga agtgcgccaa
     3061 gtgtctgaag aacaactggg agtgtcgcta ctctcccaaa accaaaaggt ctccgctgac
     3121 tagggcacat ctgacagaag tggaatcaag gctagaaaga ctggaacagc tatttctact
     3181 gatttttcct cgagaagacc ttgacatgat tttgaaaatg gattctttac aggatataaa
     3241 agcattgtta gaattcccgg gtgtcgacca gaaaaagttc aataaagtca gagttgtgag
     3301 agcactggat gctgttgctc tcccacagcc agtgggcgtt ccaaatgaaa gccaagccct
     3361 aagccagaga ttcacttttt caccaggtca agacatacag ttgattccac cactgatcaa
     3421 cctgttaatg agcattgaac cagatgtgat ctatgcagga catgacaaca caaaacctga
     3481 cacctccagt tctttgctga caagtcttaa tcaactaggc gagaggcaac ttctttcagt
     3541 agtcaagtgg tctaaatcat tgccaggttt tcgaaactta catattgatg accagataac
     3601 tctcattcag tattcttgga tgagcttaat ggtgtttggt ctaggatgga gatcctacaa
     3661 acacgtcagt gggcagatgc tgtattttgc acctgatcta atactaaatg aacagcggat
     3721 gaaagaatca tcattctatt cattatgcct taccatgtgg cagatcccac aggagtttgt
     3781 caagcttcaa gttagccaag aagagttcct ctgtatgaaa gtattgttac ttcttaatac
     3841 aattcctttg gaagggctac gaagtcaaac ccagtttgag gagatgaggt caagctacat
     3901 tagagagctc atcaaggcaa ttggtttgag gcaaaaagga gttgtgtcga gctcacagcg
     3961 tttctatcaa cttacaaaac ttcttgataa cttgcatgat cttgtcaaac aacttcatct
     4021 gtactgcttg aatacattta tccagtcccg ggcactgagt gttgaatttc cagaaatgat
     4081 gtctgaagtt attgctgggt cgacgagata tcaagcagaa ttccagtacc tgccagatac
     4141 agacgatcgt caccggattg aggagaaacg taaaaggaca tatgagacct tcaagagcat
     4201 catgaagaag agtcctttca gcggacccac cgacccccgg cctccacctc gacgcattgc
     4261 tgtgccttcc cgcagctcag cttctgtccc caagccagca ccccagccct atccctttac
     4321 gtcatccctg agcaccatca actatgatga gtttcccacc atggtgtttc cttctgggca
     4381 gatcagccag gcctcggcct tggccccggc ccctccccaa gtcctgcccc aggctccagc
     4441 ccctgcccct gctccagcca tggtatcagc tctggcccag gccccagccc ctgtcccagt
     4501 cctagcccca ggccctcctc aggctgtggc cccacctgcc cccaagccca cccaggctgg
     4561 ggaaggaacg ctgtcagagg ccctgctgca gctgcagttt gatgatgaag acctgggggc
     4621 cttgcttggc aacagcacag acccagctgt gttcacagac ctggcatccg tcgacaactc
     4681 cgagtttcag cagctgctga accagggcat acctgtggcc ccccacacaa ctgagcccat
     4741 gctgatggag taccctgagg ctataactcg cctagtgaca ggggcccaga ggccccccga
     4801 cccagctcct gctccactgg gggccccggg gctccccaat ggcctccttt caggagatga
     4861 agacttctcc tccattgcgg acatggactt ctcagccctg ctgagtcaga tcagctccta
     4921 agggggtgac gcctgccctc cccagagcac tgggttgcag gggattgaag ccctccaaaa
     4981 gcacttacgg attctggtgg ggtgtgttcc aactgccccc aactttgtgg atgtcttcct
     5041 tggagggggg agccatattt tattctttta ttgtcagtat ctgtatctct ctctcttttt
     5101 ggaggtgctt aagcagaagc attaacttct ctggaaaggg gggagctggg gaaactcaaa
     5161 cttttcccct gtcctgatgg tcagctccct tctctgtagg gaactctggg gtcccccatc
     5221 cccatcctcc agcttctggt actctcctag agacagaagc aggctggagg taaggccttt
     5281 gagcccacaa agccttatca agtgtcttcc atcatggatt cattacagct taatcaaaat
     5341 aacgccccag ataccagccc ctgtatggca ctggcattgt ccctgtgcct aacaccagcg
     5401 tttgaggggc tggccttcct gccctacaga ggtctctgcc ggctctttcc ttgctcaacc
     5461 atggctgaag gaaaccagtg caacagcact ggctctctcc aggatccgac gcgtcgagct
     5521 tatcgatacc gtcgactaaa gccaaataga aaattattca gttcctggct taagttttta
     5581 aaagtgatat tatttatttg gttgtaacca accaaaagaa tgtaaataac taatacataa
     5641 ttatgttagt tttaagttag caacaaattg attttagcta tattagctac ttggttaata
     5701 aatagaatat atttatttaa agataattgc gtttttattg tcagggagtg agtttgctta
     5761 aaaactcgtt tagatccact agttctagag cggccggatc cggaggtagc ggtggaagcg
     5821 gaggttccgg caggtaagtt attgaacaat ggcatcaaat gccttcatca tcactaccct
     5881 ttagccctta agaccccaca atgaccttac ccactcagag aaaaaagtaa atatgaaagc
     5941 ccatttgaac ttctaagctt gtcgaccatc atgatggacc agatgggtga ggtggagtac
     6001 gcgcccgggg agcccaaggg cacgccctgg cacccgcacc gcggcttcga gaccgtgacc
     6061 tacatcatgc atggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     6121 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     6181 catacgagcc gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     6241 attaattgcg ttgcgctcac tgcccgcttt ccagtcgg
//
