LOCUS       sequence-390549-        6489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insert to create split-intein Gal4[N-int] knock-in vectors, with 
            blasticidin resistance, via Drop-In cloning, released via BsmBI 
            digest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6489)
  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 6489)
  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-split-intein-Gal4[1-20]-N-int-BlastR
FEATURES             Location/Qualifiers
     source          1..6489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             89..391
                     /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     270..289
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             740..1534
                     /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             740..1534
                     /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(794..813)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1404..1423
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      1734..1743
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1740..2114
                     /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"
     rep_origin      2252..2840
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2741..2760
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2994..3011
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3128..3149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3164..3169,3170..3187,3188..3194)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3202..3218
                     /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)."
     primer_bind     3207..3229
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3226..3242
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3226..3242
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3260..3278
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3260..3277
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3396..3449
                     /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"
     protein_bind    4034..4067
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4293..4302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4299..4976
                     /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(4429..4447)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4479..4499)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4697..4716
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4891..4914
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    5097..5218
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5134..5153)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5188..5207
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5634..5655
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     protein_bind    6276..6309
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6398..6417)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6399..6417)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6424..6441)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6424..6440)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6433..6455)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
       61 ccaacagttg cgcagcctat acgtacggca gtttaaggtt tacacctata aaagagagag
      121 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgccgg ggcgacggat
      181 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
      241 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
      301 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
      361 cgccattaac ctgatgttct ggggaatata aatgtcaggc atgagattat caaaaaggat
      421 cttcacctag atccttttca cgtagaaagc cagtccgcag aaacggtgct gaccccggat
      481 gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt
      541 agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga
      601 accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg
      661 gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagctctg atcaagagac
      721 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
      781 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
      841 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
      901 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
      961 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     1021 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     1081 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     1141 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     1201 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     1261 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     1321 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     1381 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     1441 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     1501 cgccttctat cgccttcttg acgagttctt ctgaattatt aacgcttaca atttcctgat
     1561 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatcaggt ggcacttttc
     1621 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1681 cgctcatgag acaataaccc tgataaatgc ttcaataata gcacgtgagg agggccacca
     1741 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     1801 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     1861 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     1921 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     1981 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     2041 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     2101 aggagcagga ctgacacgtg ctaaaacttc atttttaatt taaaaggatc taggtgaaga
     2161 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2221 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2281 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2341 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     2401 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2461 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2521 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2581 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2641 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2701 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2761 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2821 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2881 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     2941 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3001 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     3061 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     3121 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     3181 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     3241 accatgatta cgccaagcta tttaggtgac actatagaat actcaagcta tgcatcaagc
     3301 ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt cgcccttaaa
     3361 ctgagacggg tggaagcgga ggtagcgcgg ccgccgaggg ccgcggcagc ctgctgacct
     3421 gcggcgatgt ggaggagaac cccgggcccg ctagcatgaa gctactgtct tctatcgaac
     3481 aagcatgcga tatttgccga cttaaaaagc tcaagtgcct ggatctgaag acccaggtgc
     3541 agacccccca gggcatgaag gagatcagca acatccaggt gggcgatctg gtgctgagca
     3601 acaccggcta caacgaggtg ctgaacgtgt tccccaagag caagaagaag agctacaaga
     3661 tcaccctgga ggatggcaag gagatcatct gcagcgagga gcacctgttc cccacccaga
     3721 ccggcgagat gaacatcagc ggcggcctga aggagggcat gtgcctgtac gtgaaggagt
     3781 aacctctaga gtcgactaaa gccaaataga aattattcag ttctggctta agtttttaaa
     3841 agtgatatta tttatttggt tgtaaccaac caaaagaatg taaataacta atacataatt
     3901 atgttagttt taagttagca acaaattgat tttagctata ttagctactt ggttaataaa
     3961 tagaatatat ttatttaaag ataattcgtt tttattgtca gggagtgagt ttgcttaaaa
     4021 actcgtttgg atcataactt cgtataatgt atgctatacg aagttatcgt acgggatcta
     4081 attcaattag agactaattc aattagagct aattcaatta ggatccaagc ttatcgattt
     4141 cgaaccctcg accgccggag tataaataga ggcgcttcgt ctacggagcg acaattcaat
     4201 tcaaacaagc aaagtgaaca cgtcgctaag cgaaagctaa gcaaataaac aagcgcagct
     4261 gaacaagcta aacaatcggc tcgaagccgg tcgccaccat ggcctcctcc gaggacgtca
     4321 tcaaggagtt catgcgcttc aaggtgcgca tggagggctc cgtgaacggc cacgagttcg
     4381 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     4441 tgaccaaggg cggccccctg cccttcgcct gggacatcct gtccccccag ttccagtacg
     4501 gctccaaggt gtacgtgaag caccccgccg acatccccga ctacaagaag ctgtccttcc
     4561 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     4621 cccaggactc ctccctccag gacggctcct tcatctacaa ggtgaagttc atcggcgtga
     4681 acttcccctc cgacggcccc gtaatgcaga agaagactat gggctgggag gcgtccaccg
     4741 agcgcctgta cccccgcgac ggcgtgctga agggcgagat ccacaaggcc ctgaagctga
     4801 aggacggcgg ccactacctg gtggagttca agtccatcta catggccaag aagcccgtgc
     4861 agctgcccgg ctactactac gtggactcca agctggacat cacctcccac aacgaggact
     4921 acaccatcgt ggagcagtac gagcgcgccg agggccgcca ccacctgttc ctgtaggggc
     4981 cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     5041 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     5101 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5161 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac
     5221 cgtctgtaag cagcctagaa tcccaaaaca aactggttat tgtggtaggt catttgtttg
     5281 gcagaaagaa aactcgagaa atttctctgg ccgttattcg ttattctctc ttttcttttt
     5341 gggtcgctcc ctctctgcac taatgctctc tcactctgtc acacagtaaa cggcatactg
     5401 ctctcgttgg ttcgagagag cgcgcctcga atgttcgcga aaagagcgcc ggagtataaa
     5461 tagaggcgct tcgtctacgg agcgacaatt caattcaaac aagcaaagtg aacacgtcgc
     5521 taagcgaaag ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat ctgcagtaaa
     5581 gtgcaagtta aagtgaatca attaaaagta accagcaacc aagtaaatca actgcaacta
     5641 ctgaaatctg ccaagaagta attattgaat acaagaagag aacactcaat actcgaatcc
     5701 atgtgggagt ttattcttga cacagatatt tatgatataa taactgagta agcttaacat
     5761 aaggaggaaa acaaaatgaa aaccttcaac atcagccagc aggacctgga actggtggag
     5821 gtggctaccg agaagattac gatgctctac gaggataaca agcaccacgt gggagctgcc
     5881 attcgcacca agacgggcga gattatcagt gccgtgcaca ttgaggccta catcggacgc
     5941 gtgaccgtgt gtgccgaggc cattgccatt ggcagtgccg tgagcaacgg ccagaaggat
     6001 tttgatacca ttgtggccgt gcgtcaccca tactccgatg aggtggaccg tagcatccgt
     6061 gtcgtgtccc cgtgtggaat gtgccgtgag ctgattagcg attacgcccc cgattgcttc
     6121 gtgctgattg agatgaacgg caagctggtc aagaccacca ttgaggaact gattcccctg
     6181 aagtataccc gtaactaagc ttggcaataa aaagacagaa taaaacgcac gggtgttggg
     6241 tcgtttgttc cgctgtcagg aggaaggccc atacgataac ttcgtataat gtatgctata
     6301 cgaagttatg agctccacgt ctctcgcgaa gggcgaattc tgcagatatc catcacactg
     6361 gcggccgctc gagcatgcat ctagagggcc caattcgccc tatagtgagt cgtattacaa
     6421 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     6481 tcgccttgc
//
