LOCUS       sequence-390552-        5768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insert to create ZpGal4DBD knock-ins, via Drop-In cloning, released 
            via BsmBI digest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5768)
  AUTHORS   Ewen-Campen B, Luan H, Xu J, Singh R, Joshi N, Thakkar T, Berger B, 
            White BH, Perrimon N
  TITLE     split-intein Gal4 provides intersectional genetic labeling that is 
            repressible by Gal80.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jun 13;120(24):e2304730120. doi: 
            10.1073/pnas.2304730120. Epub 2023 Jun 5.
  PUBMED    37276389
REFERENCE   2  (bases 1 to 5768)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-06-13"; volume: "120(24):e2304730120. 
            doi"; pages: " 10.1073/pnas.2304730120. Epub 2023 Jun 5"
COMMENT     Sequence Label: pDropIn-T2A-ZpGal4DBD
FEATURES             Location/Qualifiers
     source          1..5768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..34)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    23..39
                     /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(47..53,54..71,72..77))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    92..113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(230..247)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(401..989)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(481..500)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1127..1501)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     regulatory      1498..1507
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1707..2501)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(1707..2501)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1818..1837)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2428..2447
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(2850..3152)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2952..2971)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3275..3297
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3289..3306
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3290..3306
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3313..3332
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3313..3331
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3440..3493
                     /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             3686..4126
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     protein_bind    4385..4418
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4644..4653
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4650..5327
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(4780..4798)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4830..4850)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     5048..5067
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5242..5265
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    5448..5569
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5485..5504)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5539..5558
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    5599..5632
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(5731..5749)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5732..5749)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(join(5767..5768,1..15))
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(join(5767..5768,1..15))
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
       61 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
      121 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
      181 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
      241 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
      301 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
      361 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
      421 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
      481 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
      541 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
      601 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
      661 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
      721 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
      781 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
      841 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
      901 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
      961 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1021 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     1081 tcttcaccta gatcctttta aattaaaaat gaagttttag cacgtgtcag tcctgctcct
     1141 cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg
     1201 gctgctcgcc gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga
     1261 cctccgacca ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt
     1321 tgtccggcac cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca
     1381 caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact
     1441 cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca
     1501 tggtggccct cctcacgtgc tattattgaa gcatttatca gggttattgt ctcatgagcg
     1561 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1621 gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     1681 atcaggaaat tgtaagcgtt aataattcag aagaactcgt caagaaggcg atagaaggcg
     1741 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     1801 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     1861 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     1921 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gctcgccttg
     1981 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     2041 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     2101 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     2161 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     2221 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     2281 cccgtcgtgg ccagccacga tagccgcgct gcctcgtctt gcagttcatt cagggcaccg
     2341 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     2401 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     2461 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     2521 gtctcttgat cagagcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc
     2581 cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt
     2641 tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct
     2701 acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc
     2761 atccggggtc agcaccgttt ctgcggactg gctttctacg tgaaaaggat ctaggtgaag
     2821 atcctttttg ataatctcat gcctgacatt tatattcccc agaacatcag gttaatggcg
     2881 tttttgatgt cattttcgcg gtggctgaga tcagccactt cttccccgat aacggagacc
     2941 ggcacactgg ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc
     3001 gggtaaagtt cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc
     3061 atccgtcgcc ccggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg
     3121 ctctctcttt tataggtgta aaccttaaac tgccgtacgt ataggctgcg caactgttgg
     3181 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     3241 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     3301 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gccctctaga tgcatgctcg
     3361 agcggccgcc agtgtgatgg atatctgcag aattcgccct taaactgaga cgggtggaag
     3421 cggaggtagc gcggccgccg agggccgcgg cagcctgctg acctgcggcg atgtggagga
     3481 gaaccccggg cccgctagca tgctggagat ccgcgccgcc ttcctgcgcc agcgcaacac
     3541 cgccctgcgc accgaggtgg ccgagctgga gcaggaggtg cagcgcctgg agaacgaggt
     3601 gagccagtac gagacccgct acggccccct gggcggcggc aaggctagcg gaggaggtgg
     3661 tggaggtgga ggtggaggta ctagtatgaa gctgctgagt agtattgaac aagcgtgtga
     3721 catatgccga cttaagaagc tgaagtgcag caaggaaaag ccgaagtgcg ccaaatgtct
     3781 taagaacaat tgggagtgcc ggtactcgcc aaagacgaag cgaagccctt tgacacgcgc
     3841 tcatttgact gaagtggaga gtcgcctgga gcgccttgag cagttgttcc tcctcatttt
     3901 tccccgtgaa gacctcgaca tgatactgaa gatggatagt ctccaagata ttaaggccct
     3961 gctcactggc ctgtttgttc aggataacgt caacaaggat gccgtgacgg accgccttgc
     4021 ctcggtggag acagatatgc cacttacctt gcggcagcat cgcatctcgg ctaccagcag
     4081 tagtgaagaa agttccaata agggtcaacg gcaactgacg gtatcgtaag gtacctctag
     4141 agtcgactaa agccaaatag aaattattca gttctggctt aagtttttaa aagtgatatt
     4201 atttatttgg ttgtaaccaa ccaaaagaat gtaaataact aatacataat tatgttagtt
     4261 ttaagttagc aacaaattga ttttagctat attagctact tggttaataa atagaatata
     4321 tttatttaaa gataattcgt ttttattgtc agggagtgag tttgcttaaa aactcgtttg
     4381 gatcataact tcgtataatg tatgctatac gaagttatcg tacgggatct aattcaatta
     4441 gagactaatt caattagagc taattcaatt aggatccaag cttatcgatt tcgaaccctc
     4501 gaccgccgga gtataaatag aggcgcttcg tctacggagc gacaattcaa ttcaaacaag
     4561 caaagtgaac acgtcgctaa gcgaaagcta agcaaataaa caagcgcagc tgaacaagct
     4621 aaacaatcgg ctcgaagccg gtcgccacca tggcctcctc cgaggacgtc atcaaggagt
     4681 tcatgcgctt caaggtgcgc atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     4741 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     4801 gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttccagtac ggctccaagg
     4861 tgtacgtgaa gcaccccgcc gacatccccg actacaagaa gctgtccttc cccgagggct
     4921 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     4981 cctccctcca ggacggctcc ttcatctaca aggtgaagtt catcggcgtg aacttcccct
     5041 ccgacggccc cgtaatgcag aagaagacta tgggctggga ggcgtccacc gagcgcctgt
     5101 acccccgcga cggcgtgctg aagggcgaga tccacaaggc cctgaagctg aaggacggcg
     5161 gccactacct ggtggagttc aagtccatct acatggccaa gaagcccgtg cagctgcccg
     5221 gctactacta cgtggactcc aagctggaca tcacctccca caacgaggac tacaccatcg
     5281 tggagcagta cgagcgcgcc gagggccgcc accacctgtt cctgtagggg ccgcgactct
     5341 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     5401 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5461 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5521 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ccgtctgtaa
     5581 gcagcgaagg cccatacgat aacttcgtat aatgtatgct atacgaagtt atgagctcca
     5641 cgtctctcgc gaagggcgaa ttccagcaca ctggcggccg ttactagtgg atccgagctc
     5701 ggtaccaagc ttgatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta
     5761 atcatggt
//
