LOCUS       sequence-244601-        6618 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Artificial phase 2 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(2)_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             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..5617
                     /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(6113..6129)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6113..6129)
                     /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 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 acaggattat ttgtacaaga taatgtgaat aaagatgccg tcacagatag
     3301 attggcttca gtggagactg atatgcctct aacattgaga cagcatagaa taagtgcgac
     3361 atcatcatcg gaagagagta gtaacaaagg tcaaagacag ttgactgtat cgattgactc
     3421 ggcagctcat catgataact ccacaattcc gttggatttt atgcccaggg atgctcttca
     3481 tggatttgat tggtctgaag aggatgacat gtcggatggc ttgcccttcc tgaaaacgga
     3541 ccccaacaat aatgggttct ttggcgacgg ttctctctta tgtattcttc gatctattgg
     3601 ctttaaaccg gaaaattaca cgaactctaa cgttaacagg ctcccgacca tgattacgga
     3661 tagatacacg ttggcttcta gatccacaac atcccgttta cttcaaagtt atctcaataa
     3721 ttttcacccc tactgcccta tcgtgcactc accgacgcta atgatgttgt ataataacca
     3781 gattgaaatc gcgtcgaagg atcaatggca aatccttttt aactgcatat tagccattgg
     3841 agcctggtgt atagaggggg aatctactga tatagatgtt ttttactatc aaaatgctaa
     3901 atctcatttg acgagcaagg tcttcgagtc aggttccata attttggtga cagccctaca
     3961 tcttctgtcg cgatatacac agtggaggca gaaaacaaat actagctata attttcacag
     4021 cttttccata agaatggcca tatcattggg cttgaatagg gacctcccct cgtccttcag
     4081 tgatagcagc attctggaac aaagacgccg aatttggtgg tctgtctact cttgggagat
     4141 ccaattgtcc ctgctttatg gtcgatccat ccagctttct cagaatacaa tctccttccc
     4201 ttcttctgtc gacgatgtgc agcgtaccac aacaggtccc accatatatc atggcatcat
     4261 tgaaacagca aggctcttac aagttttcac aaaaatctat gaactagaca aaacagtaac
     4321 tgcagaaaaa agtcctatat gtgcaaaaaa atgcttgatg atttgtaatg agattgagga
     4381 ggtttcgaga caggcaccaa agtttttaca aatggatatt tccaccaccg ctctaaccaa
     4441 tttgttgaag gaacaccctt ggctatcctt tacaagattc gaactgaagt ggaaacagtt
     4501 gtctcttatc atttatgtat taagagattt tttcactaat tttacccaga aaaagtcaca
     4561 actagaacag gatcaaaatg atcatcaaag ttatgaagtt aaacgatgct ccatcatgtt
     4621 aagcgatgca gcacaaagaa ctgttatgtc tgtaagtagc tatatggaca atcataatgt
     4681 caccccatat tttgcctgga attgttctta ttacttgttc aatgcagtcc tagtacccat
     4741 aaagactcta ctctcaaact caaaatcgaa tgctgagaat aacgagaccg cacaattatt
     4801 acaacaaatt aacactgttc tgatgctatt aaaaaaactg gccactttta aaatccagac
     4861 ttgtgaaaaa tacattcaag tactggaaga ggtatgtgcg ccgtttctgt tatcacagtg
     4921 tgcaatccca ttaccgcata tcagttataa caatagtaat ggtagcgcca ttaaaaatat
     4981 tgtcggttct gcaactatcg cccaataccc tactcttccg gaggaaaatg tcaacaatat
     5041 cagtgttaaa tatgtttctc ctggctcagt agggccttca cctgtgccat tgaaatcagg
     5101 agcaagtttc agtgatctag tcaagctgtt atctaaccgt ccaccctctc gtaactctcc
     5161 agtgacaata ccaagaagca caccttcgca tcgctcagtc acgccttttc tagggcaaca
     5221 gcaacagctg caatcattag tgccactgac cccgtctgct ttgtttggtg gcgccaattt
     5281 taatcaaagt gggaatattg ctgatagctc attgtccttc actttcacta acagtagcaa
     5341 cggtccgaac ctcataacaa ctcaaacaaa ttctcaagcg ctttcacaac caattgcctc
     5401 ctctaacgtt catgataact tcatgaataa tgaaatcacg gctagtaaaa ttgatgatgg
     5461 taataattca aaaccactgt cacctggttg gacggaccaa actgcgtata acgcgtttgg
     5521 aatcactaca gggatgttta ataccactac aatggatgat gtatataact atctattcga
     5581 tgatgaagat accccaccaa acccaaaaaa agagtaaaat gaatcgtaga tactgaaaaa
     5641 ccccgcaagt tcacttcaac tgtgcatcgt gcaccatctc aatttctttc atttatacat
     5701 cgttttgcct tcttttatgt aactatactc ctctaagttt caatcttggc catgtaacct
     5761 ctgatctata gaatttttta aatgactaga attaatgccc atcttttttt tggacctaaa
     5821 ttcttcatga aaatatatta cgagggctta ttcagaagct tatcgatacc gtcgactaaa
     5881 gccaaataga aaattattca gttcctggct taagttttta aaagtgatat tatttatttg
     5941 gttgtaacca accaaaagaa tgtaaataac taatacataa ttatgttagt tttaagttag
     6001 caacaaattg attttagcta tattagctac ttggttaata aatagaatat atttatttaa
     6061 agataattgc gtttttattg tcagggagtg agtttgctta aaaactcgtt tagatccact
     6121 agttctagag cggccggatc cggaggtagc ggtggaagcg gaggttccgg caggtaagtt
     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
//
