LOCUS       sequence-477957-        6335 bp    DNA     circular SYN 09-JUL-2025
DEFINITION  Parental donor plasmid for scarless sfGFP-fusion knock-in fly lines.
            Red fluorescent fly eye marker to select for knock-in events, small 
            fly eye marker to select against backbone integration..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  TITLE     Perrimon Lab Plasmids 2025
REFERENCE   2  (bases 1 to 6335)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pHD-sfGFP-ScarlessDsRed-GMR-eya(shRNA)
FEATURES             Location/Qualifiers
     source          1..6335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1447..1509
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     regulatory      1904..1913
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1910..2587
                     /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(2040..2058)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2090..2110)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2308..2327
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2502..2525
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2708..2829
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2745..2764)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2799..2818
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3104..3138
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3217..3233)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3218..3234)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        3473..3711
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     3658..3679
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     primer_bind     complement(4147..4167)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4147..4165)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4186..4202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4186..4202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4199..4221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4210..4226
                     /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(4234..4240,4241..4258,4259..4264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4279..4300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4417..4434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4588..5176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4668..4687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5347..6138,6139..6207))
                     /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     5970..5989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6208..6312)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctaat
      661 tgaatttagc ggccgcgaat tcgcccttgg tggatctgga ggttccggcg gctcaggggg
      721 tagtatggtg tccaagggcg aggagctgtt caccggcgtg gtgcccatcc tggtggagct
      781 ggatggcgac gtgaacggcc acaagttcag cgtgcgcggc gagggcgagg gcgacgccac
      841 caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      901 caccctggtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgatcacat
      961 gaagcagcac gatttcttca agagcgccat gcccgagggc tacgtgcagg agcgcaccat
     1021 cagcttcaag gatgacggca cctacaagac ccgcgccgag gtgaagttcg agggcgatac
     1081 cctggtgaac cgcatcgagc tgaagggcat cgatttcaag gaggatggca acatcctggg
     1141 ccacaagctg gagtacaact tcaacagcca caacgtgtac atcaccgccg ataagcagaa
     1201 gaacggcatc aaggccaact tcaagatccg ccacaatgtg gaggatggct ccgtgcagct
     1261 ggccgatcac taccagcaga acacccccat cggcgacggc ccagtgctgc tgcccgataa
     1321 ccactacctg agcacccaga gcgtgctgtc caaggacccc aacgagaagc gcgatcacat
     1381 ggtgctgctg gagttcgtga ccgccgccgg catcaccctg ggcatggatg agctgtacaa
     1441 gattaaccct agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat
     1501 tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt
     1561 aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg
     1621 tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc ttctataaag taacaaaact
     1681 tttaggatct aattcaatta gagactaatt caattagagc taattcaatt aggatccaag
     1741 cttatcgatt tcgaaccctc gaccgccgga gtataaatag aggcgcttcg tctacggagc
     1801 gacaattcaa ttcaaacaag caaagtgaac acgtcgctaa gcgaaagcta agcaaataaa
     1861 caagcgcagc tgaacaagct aaacaatcgg ctcgaagccg gtcgccacca tggcctcctc
     1921 cgaggacgtc atcaaggagt tcatgcgctt caaggtgcgc atggagggct ccgtgaacgg
     1981 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     2041 caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca
     2101 gttccagtac ggctccaagg tgtacgtgaa gcaccccgcc gacatccccg actacaagaa
     2161 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     2221 ggtgaccgtg acccaggact cctccctcca ggacggctcc ttcatctaca aggtgaagtt
     2281 catcggcgtg aacttcccct ccgacggccc cgtaatgcag aagaagacta tgggctggga
     2341 ggcgtccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga tccacaaggc
     2401 cctgaagctg aaggacggcg gccactacct ggtggagttc aagtccatct acatggccaa
     2461 gaagcccgtg cagctgcccg gctactacta cgtggactcc aagctggaca tcacctccca
     2521 caacgaggac tacaccatcg tggagcagta cgagcgcgcc gagggccgcc accacctgtt
     2581 cctgtagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     2641 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2701 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2761 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2821 tgtatcttag atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa
     2881 catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat
     2941 catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt
     3001 gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac
     3061 ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg
     3121 cagactatct ttctagggtt aaggccggcg ggtcgggagg ttctggtggt tcaggaggtt
     3181 ccaagggcga attcgtttaa acctgcagga ctatatcgat accgtcgacc tcgagggggg
     3241 gcccggtact cgcgagcgat cgcctcgagc ccagtggaaa cccttgaaat gcctttaagt
     3301 cgagcccagt ggaaaccctt gaaatgcctt taagtcgagc ccagtggaaa cccttgaaat
     3361 gcctttaagt cgagcccagt ggaaaccctt gaaatgcctt taagtcgagc ccagtggaaa
     3421 cccttgaaat gcctttaagt cgactctagg ttaattaatc gcgaagcgag cgccggagta
     3481 taaatagagg cgcttcgtct acggagcgac aattcaattc aaacaagcaa agtgaacacg
     3541 tcgctaagcg aaagctaagc aaataaacaa gcgcagctga acaagctaaa caatctgcag
     3601 taaagtgcaa gttaaagtga atcaattaaa agtaaccagc aaccaagtaa atcaactgca
     3661 actactgaaa tctgccaaga agtaattatt gaatacaaga agagaactct gaatagggaa
     3721 ttgggaattg agatctgttc tagaaaacat cccataaaac atcccatatt cagccgctag
     3781 cagtaacatg actactacta ctacatagtt atattcaagc atatgtagta gtagtagtca
     3841 tgttgcgaaa tcaggcgaga catcggagtt gaaactaaaa ctgaattact agagtggaca
     3901 tatgcaccta ggactagagc aaactagttc tgatctgcta gacaattgtt ggcatcaggt
     3961 aggcatcaca cacgattaac aacccctaaa aatacacttt gaaaatattg aaaatatgtt
     4021 tttgtataca tttttgatat tttcaaataa tacgcagtta taaaactcat tagctaaccc
     4081 attttttctt tgcttatgct tacagattgc aaagaactag agccgcgggt accccagctt
     4141 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     4201 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4261 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4321 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4381 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4441 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4501 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4561 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4621 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4681 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4741 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4801 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4861 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4921 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4981 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5041 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5101 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5161 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5221 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5281 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5341 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5401 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5461 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5521 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5581 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5641 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5701 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5761 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5821 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5881 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5941 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6001 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6061 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6121 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6181 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6241 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6301 aggggttccg cgcacatttc cccgaaaagt gccac
//
