LOCUS       sequence-436528-        4905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Split intein version of Trojan-p65AD. Create p65AD hemi-driver lines
            from MiMIC lines, in which MiMIC cassette was inserted into Phase 2 
            coding intron. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4905)
  AUTHORS   Diao F, Vasudevan D, Heckscher ES, White BH
  TITLE     Hox gene-specific cellular targeting using split intein Trojan 
            exons.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Apr 23;121(17):e2317083121. doi: 
            10.1073/pnas.2317083121. Epub 2024 Apr 11.
  PUBMED    38602904
REFERENCE   2  (bases 1 to 4905)
  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: "2024-04-23"; volume: "121(17):e2317083121. 
            doi"; pages: " 10.1073/pnas.2317083121. Epub 2024 Apr 11"
COMMENT     Sequence Label: psiTE(2)-p65AD
FEATURES             Location/Qualifiers
     source          1..4905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..19
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3..19
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     26..45
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        26..44
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    87..156
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     CDS             588..641
                     /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             672..692
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1143..1514
                     /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             1698..1751
                     /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    2004..2073
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     complement(2119..2139)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2119..2137)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2158..2174)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2158..2174)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2171..2193)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2182..2198
                     /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(2206..2212,2213..2230,2231..2236))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2251..2272
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2389..2406)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2560..3148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2640..2659)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3319..4110,4111..4179))
                     /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     3942..3961
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4180..4284)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4311..4766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4398..4417)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4608..4629
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(4893..4905,1..10)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgacc caccggtgca
       61 tgcgcggccg cgatgtaggt cacggtctcg aagccgcggt gcgggtgcca gggcgtgccc
      121 ttgggctccc cgggcgcgta ctccacctca cccatctggt ccatcatgat gtctagaagt
      181 cgatccaaca tggcgacttg tcccatcccc ggcatgttta aatatactaa ttattcttga
      241 actaatttta atcaaccgat ttatctctct tccgcaggga atattgcctg tcctacgaca
      301 ccgagatcct gaccgtggag tacggcttcc tgcccatcgg caagatcgtg gaggagcgca
      361 tcgagtgcac cgtgtacacc gtggacaaga acggcttcgt gtacacccag cccatcgccc
      421 agtggcacaa ccgcggcgag caggaggtgt tcgagtactg cctggaggac ggctccatca
      481 tccgcgccac caaggaccac aagttcatga ccaccgacgg ccagatgctg cccatcgacg
      541 agatcttcga gcgcggcctg gacctgaagc aggtggacgg cctgcccgag ggccgcggca
      601 gcctgctgac ctgcggcgat gtggaggaga accccgggcc catggataaa gcggaattaa
      661 ttcccgagcc tccaaaaaag aagagaaagg tcgaattggg ttccacgccg atggagttcc
      721 agtacctgcc cgatacggat gaccgtcacc gtatcgaaga aaagcggaag cgaacctatg
      781 aaaccttcaa gtccatcatg aaaaagtccc ccttctcggg ccccacggac ccgcgccccc
      841 cgccccgtcg tattgcggtt ccttcgcgca gcagtgccag cgtccccaaa cccgcaccgc
      901 agccctaccc gttcacttcc tcccttagca cgattaacta tgatgagttc cccacgatgg
      961 tgttccccag tggacaaatt tcccaggcat cggcactggc tccggcccca ccgcaagtcc
     1021 tcccccaggc gcccgctccg gcaccggctc ccgcaatggt gagtgctctg gcccaggccc
     1081 ccgctccagt ccccgtgctg gcgcctggac ccccacaggc agttgcccct cctgctccga
     1141 aaccaacgca ggcgggcgaa ggaaccctga gcgaggccct cttgcagctt cagttcgatg
     1201 acgaagactt gggagccctg ctgggtaaca gcacagaccc tgccgtattc accgatctcg
     1261 catccgtgga caacagcgag tttcagcagc tcttgaatca gggaatcccg gtcgcacctc
     1321 ataccacaga gcccatgctg atggaatacc cggaggctat cacgcgactg gtgaccggcg
     1381 cacagcgacc acccgatcca gcccctgccc cactgggtgc cccgggtttg cccaatggcc
     1441 tcctcagcgg cgatgaggat ttctccagca tcgctgatat ggatttctcc gctttgctga
     1501 gccagataag ctccgctagc ggaggaggtg gtggaggtgg aggtggaggt actagtctgg
     1561 agatcgaggc cgccttcctg gagcgcgaga acaccgccct ggagacccgc gtggccgagc
     1621 tgcgccagcg cgtgcagcgc ctgcgcaacc gcgtgagcca gtaccgcacc cgctacggcc
     1681 ccctgggcgg cggcaaggaa ggacgcggat cgttgctgac gtgcggagac gtggaggaga
     1741 atccgggccc cgtgaagatc atctcccgca agtccctggg cacccagaac gtgtacgaca
     1801 tcggcgtgga gaaggaccac aacttcctgc tgaagaacgg cctggtggcc tccaactgct
     1861 tcaacaggta agttattgaa caatggcatc aaatgccttc atcatcacta ccctttagcc
     1921 cttaagaccc cacaatgacc ttacccactc agagaaaaaa gtaaatatga aagcccattt
     1981 gaacttctca tcatgatgga ccagatgggt gaggtggagt acgcgcccgg ggagcccaag
     2041 ggcacgccct ggcacccgca ccgcggcttc gagaccgtga cctacatcgg cgcgccggta
     2101 ccactagtgg ggaggttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     2161 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2221 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2281 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2401 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2461 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2521 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2581 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2641 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2701 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2761 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2821 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2881 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2941 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3001 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3061 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3121 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3181 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3241 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3301 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3361 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3421 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3481 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3541 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3601 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3661 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3721 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3781 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3841 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3901 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3961 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4021 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4081 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4141 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4201 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4261 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg
     4321 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4381 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4441 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4501 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4561 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4621 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4681 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4741 taacaaaata ttaacgctta caatttgcca ttcgccattc aggctgcgca actgttggga
     4801 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     4861 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacg
//
