LOCUS       sequence-244591-        6618 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Artificial phase 1 exon to include a spliced T2A-GAL4 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 6618)
  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 6618)
  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(1)_T2AGAL4
FEATURES             Location/Qualifiers
     source          1..6618
                     /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             2919..2972
                     /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             2973..5618
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4"
                     /product="GAL4 transcriptional activator"
                     /label=GAL4
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSIDSAAHHDNSTIPLDF
                     MPRDALHGFDWSEEDDMSDGLPFLKTDPNNNGFFGDGSLLCILRSIGFKPENYTNSNVN
                     RLPTMITDRYTLASRSTTSRLLQSYLNNFHPYCPIVHSPTLMMLYNNQIEIASKDQWQI
                     LFNCILAIGAWCIEGESTDIDVFYYQNAKSHLTSKVFESGSIILVTALHLLSRYTQWRQ
                     KTNTSYNFHSFSIRMAISLGLNRDLPSSFSDSSILEQRRRIWWSVYSWEIQLSLLYGRS
                     IQLSQNTISFPSSVDDVQRTTTGPTIYHGIIETARLLQVFTKIYELDKTVTAEKSPICA
                     KKCLMICNEIEEVSRQAPKFLQMDISTTALTNLLKEHPWLSFTRFELKWKQLSLIIYVL
                     RDFFTNFTQKKSQLEQDQNDHQSYEVKRCSIMLSDAAQRTVMSVSSYMDNHNVTPYFAW
                     NCSYYLFNAVLVPIKTLLSNSKSNAENNETAQLLQQINTVLMLLKKLATFKIQTCEKYI
                     QVLEEVCAPFLLSQCAIPLPHISYNNSNGSAIKNIVGSATIAQYPTLPEENVNNISVKY
                     VSPGSVGPSPVPLKSGASFSDLVKLLSNRPPSRNSPVTIPRSTPSHRSVTPFLGQQQQL
                     QSLVPLTPSALFGGANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     primer_bind     complement(6114..6130)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6114..6130)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    6322..6391
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     complement(6431..6451)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6431..6449)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6470..6486)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6470..6486)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6483..6505)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6494..6510
                     /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(6518..6524,6525..6542,6543..6548))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6563..6584
                     /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 tccgcaggtg
     2881 gcggaggttc cggtggaagc ggaggtagcg gcggatccga aggacgcgga agcctgttga
     2941 cgtgcggaga tgtggaagag aatccaggac cgatgaagct actgtcttct atcgaacaag
     3001 catgcgatat ttgccgactt aaaaagctca agtgctccaa agaaaaaccg aagtgcgcca
     3061 agtgtctgaa gaacaactgg gagtgtcgct actctcccaa aaccaaaagg tctccgctga
     3121 ctagggcaca tctgacagaa gtggaatcaa ggctagaaag actggaacag ctatttctac
     3181 tgatttttcc tcgagaagac cttgacatga ttttgaaaat ggattcttta caggatataa
     3241 aagcattgtt aacaggatta tttgtacaag ataatgtgaa taaagatgcc gtcacagata
     3301 gattggcttc agtggagact gatatgcctc taacattgag acagcataga ataagtgcga
     3361 catcatcatc ggaagagagt agtaacaaag gtcaaagaca gttgactgta tcgattgact
     3421 cggcagctca tcatgataac tccacaattc cgttggattt tatgcccagg gatgctcttc
     3481 atggatttga ttggtctgaa gaggatgaca tgtcggatgg cttgcccttc ctgaaaacgg
     3541 accccaacaa taatgggttc tttggcgacg gttctctctt atgtattctt cgatctattg
     3601 gctttaaacc ggaaaattac acgaactcta acgttaacag gctcccgacc atgattacgg
     3661 atagatacac gttggcttct agatccacaa catcccgttt acttcaaagt tatctcaata
     3721 attttcaccc ctactgccct atcgtgcact caccgacgct aatgatgttg tataataacc
     3781 agattgaaat cgcgtcgaag gatcaatggc aaatcctttt taactgcata ttagccattg
     3841 gagcctggtg tatagagggg gaatctactg atatagatgt tttttactat caaaatgcta
     3901 aatctcattt gacgagcaag gtcttcgagt caggttccat aattttggtg acagccctac
     3961 atcttctgtc gcgatataca cagtggaggc agaaaacaaa tactagctat aattttcaca
     4021 gcttttccat aagaatggcc atatcattgg gcttgaatag ggacctcccc tcgtccttca
     4081 gtgatagcag cattctggaa caaagacgcc gaatttggtg gtctgtctac tcttgggaga
     4141 tccaattgtc cctgctttat ggtcgatcca tccagctttc tcagaataca atctccttcc
     4201 cttcttctgt cgacgatgtg cagcgtacca caacaggtcc caccatatat catggcatca
     4261 ttgaaacagc aaggctctta caagttttca caaaaatcta tgaactagac aaaacagtaa
     4321 ctgcagaaaa aagtcctata tgtgcaaaaa aatgcttgat gatttgtaat gagattgagg
     4381 aggtttcgag acaggcacca aagtttttac aaatggatat ttccaccacc gctctaacca
     4441 atttgttgaa ggaacaccct tggctatcct ttacaagatt cgaactgaag tggaaacagt
     4501 tgtctcttat catttatgta ttaagagatt ttttcactaa ttttacccag aaaaagtcac
     4561 aactagaaca ggatcaaaat gatcatcaaa gttatgaagt taaacgatgc tccatcatgt
     4621 taagcgatgc agcacaaaga actgttatgt ctgtaagtag ctatatggac aatcataatg
     4681 tcaccccata ttttgcctgg aattgttctt attacttgtt caatgcagtc ctagtaccca
     4741 taaagactct actctcaaac tcaaaatcga atgctgagaa taacgagacc gcacaattat
     4801 tacaacaaat taacactgtt ctgatgctat taaaaaaact ggccactttt aaaatccaga
     4861 cttgtgaaaa atacattcaa gtactggaag aggtatgtgc gccgtttctg ttatcacagt
     4921 gtgcaatccc attaccgcat atcagttata acaatagtaa tggtagcgcc attaaaaata
     4981 ttgtcggttc tgcaactatc gcccaatacc ctactcttcc ggaggaaaat gtcaacaata
     5041 tcagtgttaa atatgtttct cctggctcag tagggccttc acctgtgcca ttgaaatcag
     5101 gagcaagttt cagtgatcta gtcaagctgt tatctaaccg tccaccctct cgtaactctc
     5161 cagtgacaat accaagaagc acaccttcgc atcgctcagt cacgcctttt ctagggcaac
     5221 agcaacagct gcaatcatta gtgccactga ccccgtctgc tttgtttggt ggcgccaatt
     5281 ttaatcaaag tgggaatatt gctgatagct cattgtcctt cactttcact aacagtagca
     5341 acggtccgaa cctcataaca actcaaacaa attctcaagc gctttcacaa ccaattgcct
     5401 cctctaacgt tcatgataac ttcatgaata atgaaatcac ggctagtaaa attgatgatg
     5461 gtaataattc aaaaccactg tcacctggtt ggacggacca aactgcgtat aacgcgtttg
     5521 gaatcactac agggatgttt aataccacta caatggatga tgtatataac tatctattcg
     5581 atgatgaaga taccccacca aacccaaaaa aagagtaaaa tgaatcgtag atactgaaaa
     5641 accccgcaag ttcacttcaa ctgtgcatcg tgcaccatct caatttcttt catttataca
     5701 tcgttttgcc ttcttttatg taactatact cctctaagtt tcaatcttgg ccatgtaacc
     5761 tctgatctat agaatttttt aaatgactag aattaatgcc catctttttt ttggacctaa
     5821 attcttcatg aaaatatatt acgagggctt attcagaagc ttatcgatac cgtcgactaa
     5881 agccaaatag aaaattattc agttcctggc ttaagttttt aaaagtgata ttatttattt
     5941 ggttgtaacc aaccaaaaga atgtaaataa ctaatacata attatgttag ttttaagtta
     6001 gcaacaaatt gattttagct atattagcta cttggttaat aaatagaata tatttattta
     6061 aagataattg cgtttttatt gtcagggagt gagtttgctt aaaaactcgt ttagatccac
     6121 tagttctaga gcggccggat ccggaggtag cggtggaagc ggaggttccg gaggtaagtt
     6181 attgaacaat ggcatcaaat gccttcatca tcactaccct ttagccctta agaccccaca
     6241 atgaccttac ccactcagag aaaaaagtaa atatgaaagc ccatttgaac ttctaagctt
     6301 gtcgaccatc atgatggacc agatgggtga ggtggagtac gcgcccgggg agcccaaggg
     6361 cacgccctgg cacccgcacc gcggcttcga gaccgtgacc tacatcatgc atggtaccca
     6421 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     6481 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc gggagcataa
     6541 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     6601 tgcccgcttt ccagtcgg
//
