LOCUS       sequence-173057-        6053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an EYFP gRNA ubiquitously and DsRed in eyes as a marker. 
            With attB integration site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6053)
  AUTHORS   Stern DL, Crocker J, Ding Y, Frankel N, Kappes G, Kim E, Kuzmickas 
            R, Lemire A, Mast JD, Picard S
  TITLE     Genetic and Transgenic Reagents for Drosophila simulans, D. 
            mauritiana, D. yakuba, D. santomea, and D. virilis.
  JOURNAL   G3 (Bethesda). 2017 Apr 3;7(4):1339-1347. doi: 
            10.1534/g3.116.038885.
  PUBMED    28280212
REFERENCE   2  (bases 1 to 6053)
  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: "10.1534/g3.116"; 
            journalName: "G3 (Bethesda)"; date: "2017-04-3- 3"; volume: "7"; 
            issue: "4"; pages: "1339-1347"
COMMENT     Sequence Label: p{CFD4-EYFP-3xP3::DsRed}
FEATURES             Location/Qualifiers
     source          1..6053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..681)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
                     HLFL"
     primer_bind     complement(184..204)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     596..619
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    789..923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(826..845)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     880..899
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1026..1095
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    1337..1722
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        1736..2532
                     /label=dU6-1 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-1 
                     snRNA (Port et al., 2014)"
     misc_RNA        2553..2628
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2939..2959)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2939..2957)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2978..2994)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2978..2994)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2991..3013)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3002..3018
                     /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(3026..3032,3033..3050,3051..3056))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3071..3092
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3209..3226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3380..3968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3460..3479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4139..4930,4931..4999))
                     /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     4762..4781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5000..5104)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5131..5587
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5218..5237)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5428..5449
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5713..5735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5727..5744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5728..5744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5754..5773
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5754..5772
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      join(6048..6053,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtacgct cctccaagaa cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag
       61 ggcaccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      121 ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac
      181 atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc
      241 cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
      301 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg ctgcttcatc
      361 tacaaggtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaaa
      421 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc
      481 gagatccaca aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagtcc
      541 atctacatgg ccaagaagcc cgtgcagctg cccggctact actacgtgga ctccaagctg
      601 gacatcacct cccacaacga ggactacacc atcgtggagc agtacgagcg caccgagggc
      661 cgccaccacc tgttcctgta gagatcataa tcagccatac cacatttgta gaggttttac
      721 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
      781 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
      841 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
      901 atgtatctta tcatgtctgg atcgatggcg cgcctgatat catcgatctc gaggctgcat
      961 ccaacgcgtt gggagctctc cggatcaatt cggcttcagg taccgtcgac gatgtaggtc
     1021 acggtctcga agccgcggtg cgggtgccag ggcgtgccct tgggctcccc gggcgcgtac
     1081 tccacctcac ccatctggtc catcatgatg aacgggtcga ggtggcggta gttgatcccg
     1141 gcgaacgcgc ggcgcaccgg gaagccctcg ccctcgaaac cgctgggcgc ggtggtcacg
     1201 gtgagcacgg gacgtgcgac ggcgtcggct ggtgcggata cgcggggcag cgtcagcggg
     1261 ttctcgacgg tcacggcggg catgtcgaca agccgaattg atccactaga aggcctaatt
     1321 cggtacacta gttggccacg taataagtgt gcgttgaatt tattcgcaaa aacattgcat
     1381 attttcggca aagtaaaatt ttgttgcata ccttatcaaa aaataagtgc tgcatacttt
     1441 ttagagaaac caaataattt tttattgcat acccgttttt aataaaatac attgcatacc
     1501 ctcttttaat aaaaaatatt gcatactttg acgaaacaaa ttttcgttgc atacccaata
     1561 aaagattatt atattgcata cccgttttta ataaaataca ttgcataccc tcttttaata
     1621 aagaatattg catacgttga cgaaacaaat tttcgttgca tacccaataa aagattatta
     1681 tattgcatac cttttcttgc cataccattt agccgatcaa ttgagatctg aattcatttt
     1741 caacgtcctc gatagtatag tggttagtat ccccgcctgt cacgcgggag accggggttc
     1801 aattccccgt cggggagaat ctgtgattct tttttttttt cttttacttt gttatataaa
     1861 caatttttgt tttaattgaa tctaatttgc cattgctttt aggaatctca ggcatccagc
     1921 aagcgtttgt ccgccgaatc gcccatcagt gaagaagatc ctgtggcggc tacgaaaatc
     1981 tccccggcca tgtcggcctc cacctccagc gaaaaaccca tcagcgagct ggccacctct
     2041 gtgctgaccc accgctttcc agactccacc tcctcacccg gcgaacatgg ccttggacga
     2101 atgcagttgt cgatccgcta cagcgcccag cgtcaaaaac tagacgtgac catacacaaa
     2161 atccagaaga taccacttcg cgatcccagc aatatccccg atccgtatgt taagctgtat
     2221 ctgttgcctg gacgcaccaa ggagtcgaaa cgcaagacga gcgtgatcaa ggacaactgc
     2281 aaccccgtct acgatgcatc ctttgagtac ctgatttcca ttgccgaact caggcagacg
     2341 gaactggagg tgacggtgtg cacccaaaag ggattcctat ccggcggtag tcccatcatt
     2401 ggcatggtag gtacccgaaa gcaacccctt agttacagac acagcgcgta cgtccttcgc
     2461 atccttatga ttcccaagta catattctgc aagagtacag tatatatagg aaagatatcc
     2521 gggtgaactt cgggtggtgc agatgaactt cagttttaga gctagaaata gcaagttaaa
     2581 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgcctac
     2641 ctggagcctg agagttgttc aataaaataa aaatgtttcg tttttttgct ttcgccagta
     2701 tttattattt ttcatcaata tgtattcaat ttggtatgta tttagtaatt gtaatatata
     2761 gacaatggtt ttccgttgac gtacatacat ctgacgtgtg tttatttaga cataatagtt
     2821 atgttttcac atctttttaa tgttcgctta atgcgtatgc attctagaca attgtgctcg
     2881 gcaacagtat atttgtggtg tgccaaccaa caacctgcag gagctccagc ttttgttccc
     2941 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3001 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3061 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3121 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3181 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3241 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3301 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3361 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3421 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3481 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3541 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3601 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3661 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3721 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3781 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3841 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3901 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3961 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4021 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4081 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4141 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4201 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4261 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4321 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4381 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4441 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4501 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4561 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4621 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4681 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4741 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4801 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4861 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4921 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4981 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5041 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5101 cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg
     5161 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5221 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5281 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5341 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5401 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5461 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5521 taaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5581 acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     5641 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     5701 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg
     5761 actcactata gggcgaattg ggtacaggcg cgccgggatc taattcaatt agagactaat
     5821 tcaattagag ctaattcaat taggatccaa gcttatcgat ttcgaaccct cgaccgccgg
     5881 agtataaata gaggcgcttc gtctacggag cgacaattca attcaaacaa gcaaagtgaa
     5941 cacgtcgcta agcgaaagct aagcaaataa acaagcgcag ctgaacaagc taaacaatcg
     6001 gggtaccgct agagtcgacg gtaccgcggg cccgggatcc accggtcgcc acc
//
