LOCUS       sequence-254072-        6370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Drosophila expression of sgRNA that targets Actin5c CDS close to 
            stop codon. Positive control sgRNA for CRISPaint knock-ins in 
            cultured cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6370)
  AUTHORS   Bosch JA, Colbeth R, Zirin J, Perrimon N
  TITLE     Gene Knock-Ins in Drosophila Using Homology-Independent Insertion of
            Universal Donor Plasmids.
  JOURNAL   Genetics. 2019 Nov 4. pii: genetics.119.302819. doi: 
            10.1534/genetics.119.302819.
  PUBMED    31685521
REFERENCE   2  (bases 1 to 6370)
  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. 
            2019 Nov 4. pii: genetics.119.302819. doi: 
            10.1534/genetics.119.302819."
COMMENT     Sequence Label: pCFD3-Act5c
FEATURES             Location/Qualifiers
     source          1..6370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(129..246,323..416,508..1114,1169..1302,
                     1363..1523,1597..1622))
                     /codon_start=1
                     /product="tryptophan oxygenase"
                     /label=vermilion
                     /note="selectable marker, required to synthesize the brown 
                     eye pigment in Drosophila"
                     /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE
                     EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL
                     ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY
                     SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV
                     QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF
                     YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL
                     SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
     protein_bind    1937..2006
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    2248..2633
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        2647..3067
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        3088..3163
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(3474..3494)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3474..3492)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3513..3529)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3513..3529)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3526..3548)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3537..3553
                     /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(3561..3567,3568..3585,3586..3591))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3606..3627
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3744..3761)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3915..4503)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3995..4014)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4674..5465,5466..5534))
                     /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     5297..5316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5535..5639)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5666..6122
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5753..5772)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5963..5984
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6248..6270
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6262..6279
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6263..6279
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6289..6308
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6289..6307
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gaaaaggaac acatcaaatg tgataacaaa gactaaacaa gtaattttat tacaccaaaa
       61 cgacaaaaca gtaggcagaa caaacaacgc atagccaaac attgacgaat tggataccct
      121 gccgattgtc agacactttt gttgatcagt ttcttgcgaa tggtctcgtc cagcggtgga
      181 atcgcctcgc ggggaatcag aaaagtggac agattgaaca gatccagaaa caccttgtac
      241 cgatcactga aaccaaaaaa aaacaaaggg agaacagttt gagttcattg atccccgata
      301 taatcacatc tgcgatgatc acctgagagt ggagcgcaga tattgatatc cagacgagcc
      361 accagtgccc aactgttggg atccaatcat gcgttgcacc atgatcacgt gattgtctgc
      421 ggcgggaata gaaagtattt ggttaggaaa accagtctta aacataagat atatttataa
      481 aagagtatca aagaatgcaa tacttacatc tccacttggt tattaacgag tcgatgtcca
      541 tgagcagggt gagcaactgg tgtggttggc tgaacctggg ttcatcccta tagaaggtga
      601 tcatgatggc tccctgaagg gcacgatggc taaaccggcg atccccacga cgcaccagtg
      661 catcgtgcac tgccggatca aagatggagc gatacacctc gcgtcgcttc tcaatgtcca
      721 tgaggcggta gtttttcgcc ttctccacgg gctcctccat ggcgctctgt acctgcgcct
      781 ccaggaatcg atcgacgctc tcctgaaact tggcccagaa gttgaagcca ctctcctcca
      841 gtccgggcgt cctctccagc catcgctgca ctagctccag tagcgaggga tctttctccg
      901 agttgcgaat cgagttccgc gcctcctcgt cgctaaagac atccgagtac ttctggttgt
      961 atctcacccg ctgctctgtc agaactccca gcttgttctc gatcaaacgg aactgcagcg
     1021 actgaaaacc agatgcgggt gccaggtact tgcggaagtc catgaagtct agcggggtca
     1081 tggtctccag aatgggcact tggtccacca ggagctgtac aaaggaagtt ataaacggat
     1141 tttggtaaga gattcagaaa gcactcactt ttagaatcag aaccactcgg ttcagtcgct
     1201 tgacaatctc cagcgtcttg gtttcatcga tgacctctgc atccaacatg tctcgtatgg
     1261 agtcgaactc aaagatgatc tgcttgaacc aaagctcgta ggctgtggcg aaggtactta
     1321 aatgccattg agtgttgtca tcaaagttgt aaacctactc accctggtgc gtgatgatga
     1381 acagatgctc atcgtgcacg ggtcgcttgt cctcctcgga cagcatacac tgggcatcca
     1441 gcagtttgtc cagcatcaga tactctccat agattttgcc cacttccgtg gttaatggca
     1501 ccgccgaatc atcgtgatcg tttctgtatg ggtttgaatt gaatcgcaga actgaagatc
     1561 gattggcatt cctggacagc acgtgctggt gctcacccgt ttcctgcata gggacagctc
     1621 atggtgcaca gctcagatca gatcgtgact cctcgagcgg cggatgctgg cgaactgatc
     1681 tccgccagcg gaccggagat gagaccccag cgaaccgata acagagcgag agagctccag
     1741 ttccgactga ttgcacagtc ggtgatctgg gcgatgggca ctgccagata ggctgggaat
     1801 tatcaatcac ttgaggtgaa agtgcggcgc acacaaatcc aagcttgata tcatcgatct
     1861 cgaggctgca tccaacgcgt tgggagctct ccggatcaat tcggcttcag gtaccgtcga
     1921 cgatgtaggt cacggtctcg aagccgcggt gcgggtgcca gggcgtgccc ttgggctccc
     1981 cgggcgcgta ctccacctca cccatctggt ccatcatgat gaacgggtcg aggtggcggt
     2041 agttgatccc ggcgaacgcg cggcgcaccg ggaagccctc gccctcgaaa ccgctgggcg
     2101 cggtggtcac ggtgagcacg ggacgtgcga cggcgtcggc tggtgcggat acgcggggca
     2161 gcgtcagcgg gttctcgacg gtcacggcgg gcatgtcgac aagccgaatt gatccactag
     2221 aaggcctaat tcggtacact agttggccac gtaataagtg tgcgttgaat ttattcgcaa
     2281 aaacattgca tattttcggc aaagtaaaat tttgttgcat accttatcaa aaaataagtg
     2341 ctgcatactt tttagagaaa ccaaataatt ttttattgca tacccgtttt taataaaata
     2401 cattgcatac cctcttttaa taaaaaatat tgcatacttt gacgaaacaa attttcgttg
     2461 catacccaat aaaagattat tatattgcat acccgttttt aataaaatac attgcatacc
     2521 ctcttttaat aaagaatatt gcatacgttg acgaaacaaa ttttcgttgc atacccaata
     2581 aaagattatt atattgcata ccttttcttg ccataccatt tagccgatca attgagatct
     2641 gaattctttt ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct
     2701 gtcaataaag catatttatt tatatttatt ttacaggaaa gaattccttt taaagtgtat
     2761 tttaacctat aatgaaaaac gattaaaaaa aatacataaa ataattcgaa aatttttgaa
     2821 tagcccaggt tgataaaaat tcatttcata cgttttataa cttatgcccc taagtatttt
     2881 ttgaccatag tgtttcaatt ctacattaat tttacagagt agaatgaaac gccacctact
     2941 cagccaagag gcgaaaaggt tagctcgcca agcagagagg gcgccagtgc tcactacttt
     3001 ttataattct caacttcttt ttccagactc agttcgtata tatagaccta ttttcaattt
     3061 aacgtcggcg gtgcacaatg gaggggcgtt ttagagctag aaatagcaag ttaaaataag
     3121 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg cctacctgga
     3181 gcctgagagt tgttcaataa aataaaaatg tttcgttttt ttgctttcgc cagtatttat
     3241 tatttttcat caatatgtat tcaatttggt atgtatttag taattgtaat atatagacaa
     3301 tggttttccg ttgacgtaca tacatctgac gtgtgtttat ttagacataa tagttatgtt
     3361 ttcacatctt tttaatgttc gcttaatgcg tatgcattct agacaattgt gctcggcaac
     3421 agtatatttg tggtgtgcca accaacaacc tgcaggagct ccagcttttg ttccctttag
     3481 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3541 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3601 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3661 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3721 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3781 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3841 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3901 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3961 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4021 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4081 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4141 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4201 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4261 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4321 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4381 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4441 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4501 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4561 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4621 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4681 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4741 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4801 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4861 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4921 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4981 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5041 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5101 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5161 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5221 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5281 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5341 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5401 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5461 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5521 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5581 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5641 acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5701 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5761 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5821 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5881 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5941 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     6001 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     6061 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     6121 ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     6181 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     6241 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca
     6301 ctatagggcg aattgggtac aagcttggat ttattttgtt atgttatatg tattatatgt
     6361 cagacataaa
//
