LOCUS       sequence-358579-        3990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For generation of plasmids for CRISPR-mediated 3p3 GFP knockin in 
            Drosophila.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3990)
  AUTHORS   Chen J, Luo J, Gurav AS, Chen Z, Wang Y, Montell C
  TITLE     A DREaMR system to simplify combining mutations with rescue 
            transgenes in Aedes aegypti.
  JOURNAL   Genetics. 2021 Nov 5;219(3). pii: 6368066. doi: 
            10.1093/genetics/iyab146.
  PUBMED    34740249
REFERENCE   2  (bases 1 to 3990)
  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: "Genetics.";
            date: "2021-11-5"; volume: "219(3). pii: 6368066. doi"; pages: " 
            10.1093/genetics/iyab146"
COMMENT     Sequence Label: DmU6 3p3 GFP LgRNA
FEATURES             Location/Qualifiers
     source          1..3990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      290..299
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(296..298,299..301,302..1015)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(296..298,299..301,302..1012)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(341..362)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(602..621)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     949..970
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1156..1277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1193..1212)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1320..1740
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     primer_bind     complement(1866..1882)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1866..1882)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2101..2170
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     rep_origin      complement(2257..2845)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2337..2356)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3016..3807,3808..3876))
                     /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     3639..3658
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3877..3981)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttaattaact cgcgttaaga tacattgatg agtttggaca aacctgcagg ccctttcgtc
       61 ttcaagaatt ggatctaatt caattagaga ctaattcaat tagagctaat tcaattagga
      121 tccaagctta tcgatttcga accctcgacc gccggagtat aaatagaggc gcttcgtcta
      181 cggagcgaca attcaattca aacaagcaaa gtgaacacgt cgctaagcga aagctaagca
      241 aataaacaag cgcagctgaa caagctaaac aatcggctcg aagccggtcg ccaccatggt
      301 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      361 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      421 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      481 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
      541 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      601 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
      661 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
      721 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
      781 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
      841 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
      901 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
      961 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg
     1021 cctaagcgat cgctcaggcc gcgactctag atcataatca gccataccac atttgtagag
     1081 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     1141 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1201 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1261 ctcatcaatg tatcttacct gcaggagtaa actcgtcgac ggtatcgata agcgctagct
     1321 tttttgctca cctgtgattg ctcctactca aatacaaaaa catcaaattt tctgtcaata
     1381 aagcatattt atttatattt attttacagg aaagaattcc ttttaaagtg tattttaacc
     1441 tataatgaaa aacgattaaa aaaaatacat aaaataattc gaaaattttt gaatagccca
     1501 ggttgataaa aattcatttc atacgtttta taacttatgc ccctaagtat tttttgacca
     1561 tagtgtttca attctacatt aattttacag agtagaatga aacgccacct actcagccaa
     1621 gaggcgaaaa ggttagctcg ccaagcagag agggcgccag tgctcactac tttttataat
     1681 tctcaacttc tttttccaga ctcagttcgt atatatagac ctattttcaa tttaacgtcg
     1741 gtaccggccg cgtcttagag ctatgctgga aacagcatag caagttaaga taaggctagt
     1801 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttatcgaat tcctgcagcc
     1861 cgggggatcc actagttcta gagcggcgcg cccatatgtt cggcttgtcg acatgcccgc
     1921 cgtgaccgtc gagaacccgc tgacgctgcc ccgcgtatcc gcacccgccg acgccgtcgc
     1981 acgtcccgtg ctcaccgtga ccaccgcgcc cagcggtttc gagggcgagg gcttcccggt
     2041 gcgccgcgcg ttcgccggga tcaactaccg ccacctcgac ccgttcatca tgatggacca
     2101 gatgggtgag gtggagtacg cgcccgggga gcccaagggc acgccctggc acccgcaccg
     2161 cggcttcgag accgtgacct acatcgtcga cggtaacatg tgagcaaaag gccagcaaaa
     2221 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2281 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2341 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2401 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2461 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2521 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2581 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2641 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2701 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2761 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2821 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2881 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2941 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3001 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3061 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3121 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3181 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3241 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3301 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3361 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3421 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3481 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3541 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3601 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3661 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3721 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3781 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3841 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3901 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3961 taaacaaata ggggttccgc gcacatttcc
//
