LOCUS       sequence-243784-        5428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Artificial phase 2 exon to include a spliced T2A-GAL80 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 5428)
  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 5428)
  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)_T2AGAL80
FEATURES             Location/Qualifiers
     source          1..5428
                     /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             2972..4279
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL80"
                     /product="transcriptional repressor that inhibits GAL4"
                     /label=GAL80
                     /note="GAL80 binds to and inhibits the transcriptional 
                     activation domain of GAL4."
                     /translation="MDYNKRSSVSTVPNAAPIRVGFVGLNAAKGWAIKTHYPAILQLSS
                     QFQITALYSPKIETSIATIQRLKLSNATAFPTLESFASSSTIDMIVIAIQVASHYEVVM
                     PLLEFSKNNPNLKYLFVEWALACSLDQAESIYKAAAERGVQTIISLQGRKSPYILRAKE
                     LISQGYIGDINSIEIAGNGGWYGYERPVKSPKYIYEIGNGVDLVTTTFGHTIDILQYMT
                     SSYFSRINAMVFNNIPEQELIDERGNRLGQRVPKTVPDHLLFQGTLLNGNVPVSCSFKG
                     GKPTKKFTKNLVIDIHGTKGDLKLEGDAGFAEISNLVLYYSGTRANDFPLANGQQAPLD
                     PGYDAGKEIMEVYHLRNYNAIVGNIHRLYQSISDFHFNTKKIPELPSQFVMQGFDFEGF
                     PTLMDALILHRLIESVYKSNMMGSTLNVSNISHYSL"
     protein_bind    5132..5201
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     complement(5241..5261)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5241..5259)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5280..5296)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5280..5296)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5293..5315)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5304..5320
                     /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(5328..5334,5335..5352,5353..5358))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5373..5394
                     /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 gatggactac aacaagagat cttcggtctc
     3001 aaccgtgcct aatgcagctc ccataagagt cggattcgtc ggtctcaacg cagccaaagg
     3061 atgggcaatc aagacacatt accccgccat actgcaacta tcgtcacaat ttcaaatcac
     3121 tgccttatac agtccaaaaa ttgagacttc tattgccacc attcagcgtc taaaattgag
     3181 taatgccact gcttttccca ctttagagtc atttgcatca tcttccacta tagatatgat
     3241 agtgatagct atccaagtgg ccagccatta tgaagttgtt atgcctctct tggaattctc
     3301 caaaaataat ccgaacctca agtatctttt cgtagaatgg gcccttgcat gttcactaga
     3361 tcaagccgaa tccatttata aggctgctgc tgaacgtggg gttcaaacca tcatctcttt
     3421 acaaggtcgt aaatcaccat atattttgag agcaaaagaa ttaatatctc aaggctatat
     3481 cggcgacatt aattcgatcg agattgctgg aaatggcggt tggtacggct acgaaaggcc
     3541 tgttaaatca ccaaaataca tctatgaaat cgggaacggt gtagatctgg taaccacaac
     3601 atttggtcac acaatcgata ttttacaata catgacaagt tcgtactttt ccaggataaa
     3661 tgcaatggtt ttcaataata ttccagagca agagctgata gatgagcgtg gtaaccgatt
     3721 gggccagcga gtcccaaaga cagtaccgga tcatctttta ttccaaggca cattgttaaa
     3781 tggcaatgtt ccagtgtcat gcagtttcaa aggtggcaaa cctaccaaaa aatttaccaa
     3841 aaatttggtc attgacattc acggtaccaa gggagatttg aaacttgaag gcgatgccgg
     3901 cttcgcagaa atttcaaatc tggtccttta ctacagtgga actagagcaa acgacttccc
     3961 gctagccaat ggacaacaag ctcctttaga cccggggtat gatgcaggta aagaaatcat
     4021 ggaagtatat catttacgaa attataatgc cattgtgggt aatattcatc gactgtatca
     4081 atctatctct gacttccact tcaatacaaa gaaaattcct gaattaccct cacaatttgt
     4141 aatgcaaggt ttcgatttcg aaggctttcc caccttgatg gatgctctga tattacacag
     4201 gttaatcgag agcgtttata aaagtaacat gatgggctcc acattaaacg ttagcaatat
     4261 ctcgcattat agtttataaa tgaatcgttt ttaaaataac aaatcaattg ttttataata
     4321 ttcgtacgat tctttgatta tgtaataaaa tgtgatcatt aggaagatta cgaaaaatat
     4381 aaaaaatatg agttctgtgt gtataacaaa tgctgtaaac gccacaattg tgtttgttgc
     4441 aaataaaccc atgattattt gattaaaatt gttgttttct ttgttcatag acaatagtgt
     4501 gttttgccta aacgtgtact gcataaactc catgcgagtg tatagcgagc tagtggctaa
     4561 cgcttgcccc accaaagtag attcgtcaaa atcctcaatt tcatcaccct cctccaagtt
     4621 taacatttgg ccgtcggaat taacttctaa agatgccaca taatctaata aatgaaatag
     4681 agattcaaac gtggcgtcat cgtccgtttc gaccatttcc gaaaagaact cgggcataaa
     4741 ctctatgatt tctctggacg tggtgttgtc gaaactctca aagtacgcag tcaggaacgt
     4801 gcgcgacatg tcgtcgggaa actcgcgcgg aaacatgttg ttgtaaccga acgggtccca
     4861 tagcgccaaa accaaatctg ccagcgtcaa tagaatgagc acgatgccga caatggagct
     4921 ggcttggata gcgattcgag ttaacggatc cggaggtagc ggtggaagcg gaggttccgg
     4981 caggtaagtt attgaacaat ggcatcaaat gccttcatca tcactaccct ttagccctta
     5041 agaccccaca atgaccttac ccactcagag aaaaaagtaa atatgaaagc ccatttgaac
     5101 ttctaagctt gtcgaccatc atgatggacc agatgggtga ggtggagtac gcgcccgggg
     5161 agcccaaggg cacgccctgg cacccgcacc gcggcttcga gaccgtgacc tacatcatgc
     5221 atggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     5281 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5341 gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5401 ttgcgctcac tgcccgcttt ccagtcgg
//
