LOCUS       sequence-331636-        8884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For germline transformation. Expresses white exon 2 sgRNA and DsRed 
            marker in all cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8884)
  AUTHORS   Kandul NP, Belikoff EJ, Liu J, Buchman A, Li F, Yamamoto A, Yang T, 
            Shriner I, Scott MJ, Akbari OS
  TITLE     Genetically Encoded CRISPR Components Yield Efficient Gene Editing 
            in the Invasive Pest Drosophila suzukii.
  JOURNAL   CRISPR J. 2021 Oct;4(5):739-751. doi: 10.1089/crispr.2021.0032.
  PUBMED    34661429
REFERENCE   2  (bases 1 to 8884)
  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"; doi: 
            "10.1089/crispr.2021"; journalName: "CRISPR J"; date: "2021-10"; 
            volume: "4"; issue: "5"; pages: "739-751"
COMMENT     Sequence Label: pB[U6-3 wex2 PUb-DsRed]
FEATURES             Location/Qualifiers
     source          1..8884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     236..253
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    370..391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(406..411,412..429,430..436)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    444..460
                     /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)."
     primer_bind     449..471
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     468..484
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     468..484
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   975..1009
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1292..1308)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1293..1309)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1333..1352)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1334..1352)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1365..1785
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        1805..1880
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    2125..2510
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     CDS             complement(3219..3896)
                     /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
                     VTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEPSTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(3281..3304)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3479..3498)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3696..3716
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3748..3766
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3893..3902
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6048..6147
                     /label=phage phi-C31 attP
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attachment site of phage phi-C31"
     repeat_region   6411..6473
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6603..6620)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6603..6619)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6612..6634)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6761..7216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6848..6867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7058..7079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7242..7346
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7347..7415,7416..8207)
                     /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(7565..7584)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8378..8884,1..82)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     join(8867..8884,1..2)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
       61 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      121 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      181 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      241 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
      301 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      361 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
      421 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
      481 tgaccatgat tacgccaagc gcgcccgccg ggtaactcac ggggtatcca tgtccatttc
      541 tgcggcatcc agccaggata cccgtcctcg ctgacgtaat atcccagcgc cgcaccgctg
      601 tcattaatct gcacaccggc acggcagttc cggctgtcgc cggtattgtt cgggttgctg
      661 atgcgcttcg ggctgaccat ccggaactgt gtccggaaaa gccgcgacga actggtatcc
      721 caggtggcct gaacgaacag ttcaccgtta aaggcgtgca tggccacacc ttcccgaatc
      781 atcatggtaa acgtgcgttt tcgctcaacg tcaatgcagc agcagtcatc ctcggcaaac
      841 tctttccatg ccgcttcaac ctcgcgggaa aaggcacggg cttcttcctc cccgatgccc
      901 agatagcgcc agcttgggcg atgactgagc cggaaaaaag acccgacgat atgatcctga
      961 tgcagctaga ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa
     1021 tattgctctc tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc
     1081 gccgcttgga gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac
     1141 tataacgacc gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt
     1201 aactcatacg ataattatat tgttatttca tgttctactt acgtgataac ttattatata
     1261 tatattttct tgttatagat atcaagctta tcgataccgt cgacctcgag ggggggcccg
     1321 gtacccaatt cgccctatag tgagtcgtat taagatctga attctttttt gctcacctgt
     1381 gattgctcct actcaaatac aaaaacatca aattttctgt caataaagca tatttattta
     1441 tatttatttt acaggaaaga attcctttta aagtgtattt taacctataa tgaaaaacga
     1501 ttaaaaaaaa tacataaaat aattcgaaaa tttttgaata gcccaggttg ataaaaattc
     1561 atttcatacg ttttataact tatgccccta agtatttttt gaccatagtg tttcaattct
     1621 acattaattt tacagagtag aatgaaacgc cacctactca gccaagaggc gaaaaggtta
     1681 gctcgccaag cagagagggc gccagtgctc actacttttt ataattctca acttcttttt
     1741 ccagactcag ttcgtatata tagacctatt ttcaatttaa cttcggcggt gaaggagtcc
     1801 agtcgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     1861 aagtggcacc gagtcggtgc ttttttgcct acctggagcc tgagagttgt tcaataaaat
     1921 aaaaatgttt cgtttttttg ctttcgccag tatttattat ttttcatcaa tatgtattca
     1981 atttggtatg tatttagtaa ttgtaatata tagacaatgg ttttccgttg acgtacatac
     2041 atctgacgtg tgtttattta gacataatag ttatgttttc acatcttttt aatgttcgct
     2101 taatgcgtat gcattctagt tggccacgta ataagtgtgc gttgaattta ttcgcaaaaa
     2161 cattgcatat tttcggcaaa gtaaaatttt gttgcatacc ttatcaaaaa ataagtgctg
     2221 catacttttt agagaaacca aataattttt tattgcatac ccgtttttaa taaaatacat
     2281 tgcataccct cttttaataa aaaatattgc atactttgac gaaacaaatt ttcgttgcat
     2341 acccaataaa agattattat attgcatacc cgtttttaat aaaatacatt gcataccctc
     2401 ttttaataaa gaatattgca tacgttgacg aaacaaattt tcgttgcata cccaataaaa
     2461 gattattata ttgcatacct tttcttgcca taccatttag ccgatcaatt gagatctagg
     2521 cgcgccaagg ccggccgtta actcgaatcg ctatccaagc cagctccatt gtcggcatcg
     2581 tgctcattct attgacgctg gcagatttgg ttttggcgct atgggacccg ttcggttaca
     2641 acaacatgtt tccgcgcgag tttcccgacg acatgtcgcg cacgttcctg actgcgtact
     2701 ttgagagttt cgacaacacc acgtccagag aaatcataga gtttatgccc gagttctttt
     2761 cggaaatggt cgaaacggac gatgacgcca cgtttgaatc tctatttcat ttattagatt
     2821 atgtggcatc tttagaagtt aattccgacg gccaaatgtt aaacttggag gagggtgatg
     2881 aaattgagga ttttgacgaa tctactttgg tggggcaagc gttagccact agctcgctat
     2941 acactcgcat ggagtttatg cagtacacgt ttaggcaaaa cacactattg tctatgaaca
     3001 aagaaaacaa caattttaat caaataatca tgggtttatt tgcaacaaac acaattgtgg
     3061 cgtttacagc atttgttata cacacagaac tcatattttt tatatttttc gtaatcttcc
     3121 taatgatcac attttattac ataatcaaag aatcgtacga atattataaa acaattgatt
     3181 tgttatttta aaaacgattc attctagagt cgcggccgct acaggaacag gtggtggcgg
     3241 ccctcggtgc gctcgtactg ctccacgatg gtgtagtcct cgttgtggga ggtgatgtcc
     3301 agcttggagt ccacgtagta gtagccgggc agctgcacgg gcttcttggc catgtagatg
     3361 gacttgaact ccaccaggta gtggccgccg tccttcagct tcagggcctt gtggatctcg
     3421 cccttcagca cgccgtcgcg ggggtacagg cgctcggtgg agggctccca gcccatagtc
     3481 ttcttctgca ttacggggcc gtcggagggg aagttcacgc cgatgaactt caccttgtag
     3541 atgaagcagc cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg
     3601 aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttctt gtagtcgggg
     3661 atgtcggcgg ggtgcttcac gtacaccttg gagccgtact ggaactgggg ggacaggatg
     3721 tcccaggcga agggcagggg gccgcccttg gtcaccttca gcttggcggt ctgggtgccc
     3781 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc
     3841 tccatgcgca ccttgaagcg catgaactcc ttgatgacgt cctcggagga ggccatggtg
     3901 gcgactagtg gatccccgat ctgcattttg gattattctg cgggcagaaa atagagatgt
     3961 ggaaaattag tacgaaatca aatgagtttc gttgaaatta caaaactatt gaaactaact
     4021 tcctggctgg ggaataaaaa tgggaaactt atttatcgac gccaactttg ttgagaaacc
     4081 cctattaacc ctctacgaat attggaacaa aggaaagcga agaaacaggg aacaaagggt
     4141 agttgagaaa cctttgttcc gttgcttcgt catcgttttc gtgttgcgag tgtgtgcatg
     4201 tatatataca cagctgaaat gcatgcatac acattatttt gtgtgtatat ggtgacgtca
     4261 caactacaag caataagaaa ttttcagacg tggctttgtt taagcaacct actctatttc
     4321 agctaaaaat aagtggattt cgttggtaaa atacttcaat taactaaaca actaactaat
     4381 aacatgcaca caaatgctcg agtgcgttcg tgatttctcg aattttcaaa tgcgtcactg
     4441 cgaatttcac aatttgccaa taaatcttgg cgaaaatcaa cacgcaagtt ttatttatag
     4501 atttgtttgc gttttgatgc caattcattg ggaaaacaag atgcgtggct gccaatttct
     4561 tattttgtaa ttacgtagag cgttgaataa aaaaaaaatg gccgaacaaa gaccttgaaa
     4621 tgcagttttt cttgaaatta ctcaacgtct tgttgctctt attactaatt ggtaacagcg
     4681 agttaaaaac ttacgtttct tgtgactttc gagaatgttc ttttaattgt actttaatca
     4741 ccaacaatta agtataaatt tttcgctgat tgcgctttac tttctacttg tacttgctgc
     4801 tgcaaatgtc aattggtttt gaaggcgacc gttcgcgaac gctgtttata taccttcggt
     4861 gtccgttgaa aatcactaaa aaataccgta gtgttcgtaa cactttagta cagagaaaaa
     4921 aaattgtgcc gaaatgtttt tgatacgtac gaataccttg tattaaaatt ttttatgatt
     4981 tctgtgtatc actttttttt tgtgtttttc gtttaaactc accacagtac aaaacaataa
     5041 aatattttta agacaatttc aaattgagac ctttctcgta ctgacttgac cggctgaatg
     5101 aggatttcta cctagacgac ctacttctta ccatgacatt gaatgcaatg ccacctttga
     5161 tctaaactta caaaagtcca aggcttgtta ggattggtgt tttggaattt aagacccttg
     5221 ttagtttgct tttgaaatag cactgtcttc tctaccggct ataattttga aactcgcagc
     5281 ttgactggaa atttaaaaag taattctgtg taggtaaagg gtgttttaaa agtgtgatgt
     5341 gttgagcgtt gcggcaacga ctgctattta tgtatatatt ttcaaaactt attgtttttg
     5401 aagtgtttta aatggagcta tctggcaacg ctgcgcataa tcttacacaa gcttttctta
     5461 atccattttt aagtgaaatt tgtttatact ctttcggcaa ataattgtta aatcgcttta
     5521 agtgggctta catctggata agtaatgaaa acctgcatat tataatatta aaacatataa
     5581 tccactgtgc tttccccgtg tgtggccata tacctaaaaa agtttatttt cgcagagccc
     5641 cgcacggtca cactacggtt cggcgatttt cgattttgga cagtactgat tgcaagcgca
     5701 ccgaaagcaa aatggagctg gagattttga acgcgaagaa cagcaagccg tacggcaagg
     5761 tgaaggtgcc ctccggcgcc acgcccatcg gcgatctgcg cgccctaatt cacaagaccc
     5821 tgaagcagac cccacacgcg aatcgccagt cgcttcgtct ggaactgaag ggcaaaagcc
     5881 tgaaagatac ggacacattg gaatctctgt cgctgcgttc cggcgacaag atcgggtacc
     5941 gtcgactgca gaattcgaag cttgagctcg agatcccccg ggctgtacta gtcgcgctcg
     6001 cgcgactgac ggtcgtaagc acccgcgtac gtgtccaccc cggtcacaac cccttgtgtc
     6061 atgtcggcga ccctacgccc ccaactgaga gaactcaaag gttaccccag ttggggcact
     6121 actcccgaaa accgcttctg acctgggaaa acgtgaagcc ccggggcatc cgctgagggt
     6181 tgccgccggg gcttcggtgt gtccgtcagt actagtcccg ggctgcagga attcgataaa
     6241 agttttgtta ctttatagaa gaaattttga gtttttgttt tttttaataa ataaataaac
     6301 ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta
     6361 atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa
     6421 ttttacacat gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaatct
     6481 agctgcgtgt tctgcagcgt gtcgagcatc ttcatctgct ccatcacgct gtaaaacaca
     6541 tttgcaccgc gagtctgccc gtcctccacg ggttcaaaaa cgtgaatgaa cgaggcgcgc
     6601 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     6661 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     6721 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     6781 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     6841 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     6901 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     6961 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7021 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     7081 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     7141 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     7201 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     7261 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     7321 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     7381 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     7441 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     7501 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     7561 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     7621 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     7681 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     7741 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     7801 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     7861 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     7921 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     7981 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     8041 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     8101 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     8161 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     8221 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     8281 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     8341 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     8401 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     8461 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     8521 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     8581 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     8641 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     8701 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     8761 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     8821 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     8881 atct
//
