LOCUS       sequence-336460-        5777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For scarless insertion of 3x127D01 NanoTag into C-term; Marked by 
            the visible marker 3xP3-DsRed flanked by piggyBac termini..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5777)
  AUTHORS   Xu J, Kim AR, Cheloha RW, Fischer FA, Li JSS, Feng Y, Stoneburner E,
            Binari R, Mohr SE, Zirin J, Ploegh HL, Perrimon N
  TITLE     Protein visualization and manipulation in Drosophila through the use
            of epitope tags recognized by nanobodies.
  JOURNAL   Elife. 2022 Jan 25;11. pii: 74326. doi: 10.7554/eLife.74326.
  PUBMED    35076390
REFERENCE   2  (bases 1 to 5777)
  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: "Elife."; 
            date: "2022-01-25"; volume: "11. pii: 74326. doi"; pages: " 
            10.7554/eLife.74326"
COMMENT     Sequence Label: pScarlessHD-C-3x127D01-DsRed
FEATURES             Location/Qualifiers
     source          1..5777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(894..962,963..1754)
                     /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     complement(1112..1131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(2004..2798)
                     /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(2004..2798)
                     /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(2115..2134)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2725..2744
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3147..3449)
                     /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(3249..3268)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3572..3594
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3586..3603
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3587..3603
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3610..3629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3610..3628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   3794..3856
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     regulatory      4251..4260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4257..4934
                     /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(4387..4405)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4437..4457)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4655..4674
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4849..4872
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    5055..5176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5092..5111)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5146..5165
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5451..5485
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5518..5536)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5557..5573)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5557..5573)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5570..5592)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5581..5597
                     /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(5605..5611,5612..5629,5630..5635))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5650..5671
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      901 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      961 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1021 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1081 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1141 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1201 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1261 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1321 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1381 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1441 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1501 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1561 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1621 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1681 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1741 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1801 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     1981 aggaaattgt aagcgttaat aattcagaag aactcgtcaa gaaggcgata gaaggcgatg
     2041 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
     2101 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
     2161 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
     2221 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgct cgccttgagc
     2281 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
     2341 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
     2401 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
     2461 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat
     2521 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
     2581 gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac
     2641 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     2701 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     2761 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc
     2821 tcttgatcag agcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag
     2881 tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg
     2941 cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc
     3001 tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc
     3061 cggggtcagc accgtttctg cggactggct ttctacgtga aaaggatcta ggtgaagatc
     3121 ctttttgata atctcatgcc tgacatttat attccccaga acatcaggtt aatggcgttt
     3181 ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc
     3241 acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg
     3301 taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg gatcaccatc
     3361 cgtcgccccg gcgtgtcaat aatatcactc tgtacatcca caaacagacg ataacggctc
     3421 tctcttttat aggtgtaaac cttaaactgc cgtacgtata ggctgcgcaa ctgttgggaa
     3481 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     3541 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     3601 agtgaattgt aatacgactc actatagggc gaattgaatt tagcggccgc gaattcggag
     3661 gtggcagcgg ctccggcagt tttgaggact tttggaaagg cgaggatggt agcggaagtt
     3721 tcgaagactt ctggaaggga gaggatggat ccggaagctt tgaagacttt tggaaaggag
     3781 aagactagat taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc
     3841 gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat
     3901 agatattaag ttttattata tttacactta catactaata ataaattcaa caaacaattt
     3961 atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa
     4021 caaaactttt aggatctaat tcaattagag actaattcaa ttagagctaa ttcaattagg
     4081 atccaagctt atcgatttcg aaccctcgac cgccggagta taaatagagg cgcttcgtct
     4141 acggagcgac aattcaattc aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc
     4201 aaataaacaa gcgcagctga acaagctaaa caatcggctc gaagccggtc gccaccatgg
     4261 cctcctccga ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg
     4321 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     4381 agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt
     4441 ccccccagtt ccagtacggc tccaaggtgt acgtgaagca ccccgccgac atccccgact
     4501 acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     4561 gcggcgtggt gaccgtgacc caggactcct ccctccagga cggctccttc atctacaagg
     4621 tgaagttcat cggcgtgaac ttcccctccg acggccccgt aatgcagaag aagactatgg
     4681 gctgggaggc gtccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc
     4741 acaaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag tccatctaca
     4801 tggccaagaa gcccgtgcag ctgcccggct actactacgt ggactccaag ctggacatca
     4861 cctcccacaa cgaggactac accatcgtgg agcagtacga gcgcgccgag ggccgccacc
     4921 acctgttcct gtagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     4981 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     5041 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5101 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5161 tcatcaatgt atcttagata tctataacaa gaaaatatat atataataag ttatcacgta
     5221 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
     5281 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
     5341 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
     5401 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
     5461 ttttacgcag actatctttc tagggttaag aattcgttta aacctgcagg actagtccct
     5521 ttagtgaggg ttaattctga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5581 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5641 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5701 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5761 tttgcgtatt gggcgct
//
