LOCUS       sequence-390554-        6197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insert to create p65Zp 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 6197)
  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 6197)
  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-p65Zp
FEATURES             Location/Qualifiers
     source          1..6197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(306..608)
                     /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(408..427)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     731..753
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     745..762
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     746..762
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     769..788
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        769..787
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(877..910)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    940..1061
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(951..970)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1005..1024
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1182..1859)
                     /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(1244..1267)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1442..1461)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1659..1679
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1711..1729
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1856..1865
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2091..2124)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2566..2937)
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     CDS             complement(3388..3408)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3445..3498)
                     /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"
     promoter        complement(3616..3634)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3617..3634)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3652..3668)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3652..3668)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3665..3687)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3676..3692
                     /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(3700..3706,3707..3724,3725..3730))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3745..3766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3883..3900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4054..4642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4134..4153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4780..5154)
                     /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      5151..5160
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5360..6154)
                     /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(5360..6154)
                     /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(5471..5490)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6081..6100
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 ctgcgccatc agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca
       61 accttaccag agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc
      121 cagtctagct atcgccatgt aagcccactg caagctacct gctttctctt tgcgcttgcg
      181 ttttcccttg tccagatagc ccagtagctg acattcatcc ggggtcagca ccgtttctgc
      241 ggactggctt tctacgtgaa aaggatctag gtgaagatcc tttttgataa tctcatgcct
      301 gacatttata ttccccagaa catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg
      361 ctgagatcag ccacttcttc cccgataacg gagaccggca cactggccat atcggtggtc
      421 atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg ggagacttta
      481 tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcgccccgg cgtgtcaata
      541 atatcactct gtacatccac aaacagacga taacggctct ctcttttata ggtgtaaacc
      601 ttaaactgcc gtacgtatag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
      661 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
      721 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca
      781 ctatagggcg aattgggccc tctagatgca tgctcgagcg gccgccagtg tgatggatat
      841 ctgcagaatt cgcccttcgc gagagacgtg gagctcataa cttcgtatag catacattat
      901 acgaagttat cgtatgggcc ttcgctgctt acagacggtt aagatacatt gatgagtttg
      961 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     1021 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     1081 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
     1141 acaaatgtgg tatggctgat tatgatctag agtcgcggcc cctacaggaa caggtggtgg
     1201 cggccctcgg cgcgctcgta ctgctccacg atggtgtagt cctcgttgtg ggaggtgatg
     1261 tccagcttgg agtccacgta gtagtagccg ggcagctgca cgggcttctt ggccatgtag
     1321 atggacttga actccaccag gtagtggccg ccgtccttca gcttcagggc cttgtggatc
     1381 tcgcccttca gcacgccgtc gcgggggtac aggcgctcgg tggacgcctc ccagcccata
     1441 gtcttcttct gcattacggg gccgtcggag gggaagttca cgccgatgaa cttcaccttg
     1501 tagatgaagg agccgtcctg gagggaggag tcctgggtca cggtcaccac gccgccgtcc
     1561 tcgaagttca tcacgcgctc ccacttgaag ccctcgggga aggacagctt cttgtagtcg
     1621 gggatgtcgg cggggtgctt cacgtacacc ttggagccgt actggaactg gggggacagg
     1681 atgtcccagg cgaagggcag ggggccgccc ttggtcacct tcagcttggc ggtctgggtg
     1741 ccctcgtagg ggcggccctc gccctcgccc tcgatctcga actcgtggcc gttcacggag
     1801 ccctccatgc gcaccttgaa gcgcatgaac tccttgatga cgtcctcgga ggaggccatg
     1861 gtggcgaccg gcttcgagcc gattgtttag cttgttcagc tgcgcttgtt tatttgctta
     1921 gctttcgctt agcgacgtgt tcactttgct tgtttgaatt gaattgtcgc tccgtagacg
     1981 aagcgcctct atttatactc cggcggtcga gggttcgaaa tcgataagct tggatcctaa
     2041 ttgaattagc tctaattgaa ttagtctcta attgaattag atcccgtacg ataacttcgt
     2101 atagcataca ttatacgaag ttatgatcca aacgagtttt taagcaaact cactccctga
     2161 caataaaaac gaattatctt taaataaata tattctattt attaaccaag tagctaatat
     2221 agctaaaatc aatttgttgc taacttaaaa ctaacataat tatgtattag ttatttacat
     2281 tcttttggtt ggttacaacc aaataaataa tatcactttt aaaaacttaa gccagaactg
     2341 aataatttct atttggcttt agtcgactct agaggtacct tacttgccgc cgcccagggg
     2401 gccgtagcgg gtgcggtact ggctcacgcg gttgcgcagg cgctgcacgc gctggcgcag
     2461 ctcggccacg cgggtctcca gggcggtgtt ctcgcgctcc aggaaggcgg cctcgatctc
     2521 cagactagta cctccacctc cacctccacc acctcctccg ctagcggagc ttatctggct
     2581 cagcaaagcg gagaaatcca tatcagcgat gctggagaaa tcctcatcgc cgctgaggag
     2641 gccattgggc aaacccgggg cacccagtgg ggcaggggct ggatcgggtg gtcgctgtgc
     2701 gccggtcacc agtcgcgtga tagcctccgg gtattccatc agcatgggct ctgtggtatg
     2761 aggtgcgacc gggattccct gattcaagag ctgctgaaac tcgctgttgt ccacggatgc
     2821 gagatcggtg aatacggcag ggtctgtgct gttacccagc agggctccca agtcttcgtc
     2881 atcgaactga agctgcaaga gggcctcgct cagggttcct tcgcccgcct gcgttggttt
     2941 cggagcagga ggggcaactg cctgtggggg tccaggcgcc agcacgggga ctggagcggg
     3001 ggcctgggcc agagcactca ccattgcggg agccggtgcc ggagcgggcg cctgggggag
     3061 gacttgcggt ggggccggag ccagtgccga tgcctgggaa atttgtccac tggggaacac
     3121 catcgtgggg aactcatcat agttaatcgt gctaagggag gaagtgaacg ggtagggctg
     3181 cggtgcgggt ttggggacgc tggcactgct gcgcgaagga accgcaatac gacggggcgg
     3241 ggggcgcggg tccgtggggc ccgagaaggg ggactttttc atgatggact tgaaggtttc
     3301 ataggttcgc ttccgctttt cttcgatacg gtgacggtca tccgtatcgg gcaggtactg
     3361 gaactccatc ggcgtggatc ccaattcgac ctttctcttc ttttttggag gctcgggaat
     3421 taattccgct ttatccatgc tagcgggccc ggggttctcc tccacatcgc cgcaggtcag
     3481 caggctgccg cggccctcgg cggccgcgct acctccgctt ccacccgtct cagtttaagg
     3541 gcgaattcca gcacactggc ggccgttact agtggatccg agctcggtac caagcttgat
     3601 gcatagcttg agtattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct
     3661 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3721 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3781 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3841 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3901 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3961 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4021 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4081 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4141 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4201 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4261 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4321 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4381 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4441 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4501 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4561 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4621 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4681 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4741 ctagatcctt ttaaattaaa aatgaagttt tagcacgtgt cagtcctgct cctcggccac
     4801 gaagtgcacg cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc
     4861 gccgatctcg gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga
     4921 ccactcggcg tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg
     4981 caccacctgg tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc
     5041 gaagtcgtcc tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc
     5101 tccggcgacg tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggtggc
     5161 cctcctcacg tgctattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5221 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5281 gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga
     5341 aattgtaagc gttaataatt cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     5401 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     5461 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     5521 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     5581 aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgctcgcc ttgagcctgg
     5641 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     5701 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     5761 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     5821 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     5881 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     5941 tggccagcca cgatagccgc gctgcctcgt cttgcagttc attcagggca ccggacaggt
     6001 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     6061 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     6121 gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt
     6181 gatcagagct tgatccc
//
