LOCUS       sequence-213590-        6759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of fly mutant PP4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6759)
  TITLE     Di Giovanni Lab neuroscience plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6759)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pcDNA3. 3xFlag:PP4-19c[D85NH115N]
FEATURES             Location/Qualifiers
     source          1..6759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        66..445
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        446..649
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     600..620
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     694..713
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        694..712
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      754..763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             763..828
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    838..862
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1790..1814)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    1864..1998
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(2143..2161)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2144..2161)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    2187..2411
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2457..2885
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2544..2563)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2754..2775
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2894..2914)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2899..3228
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3079..3214
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3141..3160
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3295..4089
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3349..3368)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3959..3978
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4263..4384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4433..4449)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4433..4449)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4446..4468)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4457..4473
                     /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(4481..4487,4488..4505,4506..4511))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4526..4547
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4664..4681)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4835..5423)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4915..4934)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5594..6385,6386..6454))
                     /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     6217..6236
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6455..6559)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6634..6653)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
       61 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      121 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      181 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      241 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      301 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      361 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      421 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      481 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      541 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      601 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
      661 tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca
      721 agcttggtac cactgcagtg aattcggagc tccgccacca tggactacaa agaccatgac
      781 ggtgattata aagatcatga catcgattac aaggatgacg atgacaagca ccggtataca
      841 agtttgtaca aaaaagcagg cttaatgtcc gactacagcg acctggaccg acagatcgag
      901 caactgaagc gctgcgagat catcaaggag aacgaggtga aggccctgtg cgccaaagcc
      961 cgcgagatcc tggtggagga gggcaacgtg cagcgtgtgg actcgccagt gaccgtgtgc
     1021 ggcgacatcc acggtcagtt ctacgacctg aaggagctgt tcaaggttgg cggcgatgtg
     1081 cccgagaaga actacctgtt tatgggcgat ttcgtgaacc gcggctacta tagtgtggag
     1141 acgtttctgc tgctgttggc gctgaaggtg cgctatccgg accgaatcac gctgatccgg
     1201 ggcaacaacg agtcgcgcca aatcacacag gtctacggct tttacgatga gtgcctgcgc
     1261 aagtacggat cgacggccgt gtggcggtac tgcacggaga tctttgacta cctcagcctt
     1321 tcggcgatca tcgacggcaa aatattctgc gtgcacggcg ggctgtcgcc gtccatccag
     1381 tacctcgacc agattcgcag catcgaccgg aagcaggagg tgccgcacga cgggcccatg
     1441 tgcgacctgc tttggagcga tccggaggac cagactggct ggggcgtgtc gccgcgcggc
     1501 gctggctatc tctttggctc ggacgtggtc tcccagttca accgcacgaa cgatatcgac
     1561 atgatttgcc gtgcacacca gttggtaatg gagggcttca agtggcactt caacgaaacc
     1621 gtcctgaccg tctggtcggc acccaattac tgctatcgct gcggcaatgt agctgccatc
     1681 ctggagctga atgagtacct gcaccgcgac ttcgtcatct ttgaggcagc tccgcaagag
     1741 agtcgcggca ttccctcgaa gaagcctcag gcggactact ttctctaata cccagctttc
     1801 ttgtacaaag tggtgacgta agctagttta aacccgctga tcagcctcga ctgtgccttc
     1861 taagatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     1921 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     1981 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     2041 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
     2101 gatcagtcga ggccgctcga gcatgcatct agagggccct attctatagt gtcacctaaa
     2161 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     2221 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2281 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2341 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2401 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     2461 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2521 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2581 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2641 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2701 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2761 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2821 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2881 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2941 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3001 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3061 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3121 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     3181 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     3241 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     3301 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3361 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3421 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3481 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3541 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3601 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3661 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3721 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3781 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     3841 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3901 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3961 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4021 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4081 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4141 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4201 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4261 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4321 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4381 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4441 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4501 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4561 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4621 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4681 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4741 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4801 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4861 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4921 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4981 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5041 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5101 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5161 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5221 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5281 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5341 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5401 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5461 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5521 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5581 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5641 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5701 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5761 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5821 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5881 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5941 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6001 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6061 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6121 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6181 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6241 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6301 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6361 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6421 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6481 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6541 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     6601 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6661 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     6721 ttaagctaca acaaggcaag gcttgaccga caattgcat
//
