LOCUS       sequence-261570-        6815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA vector for targeting near the attP40 locus, use with 
            pHD-3XP3-dsRed-DattP-CRISPR-donor-attP40 based constructs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6815)
  AUTHORS   Isaacman-Beck J, Paik KC, Wienecke CFR, Yang HH, Fisher YE, Wang IE,
            Ishida IG, Maimon G, Wilson RI, Clandinin TR
  TITLE     SPARC enables genetic manipulation of precise proportions of cells.
  JOURNAL   Nat Neurosci. 2020 Sep;23(9):1168-1175. doi: 
            10.1038/s41593-020-0668-9. Epub 2020 Jul 20.
  PUBMED    32690967
REFERENCE   2  (bases 1 to 6815)
  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: 
            "10.1038/s41593-020-0668-9"; journalName: "Nat Neurosci"; date: 
            "2020-09"; volume: "23"; issue: "9"; pages: "1168-1175"
COMMENT     Sequence Label: pCFD5-U6-3-t-attP40
FEATURES             Location/Qualifiers
     source          1..6815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        12..432
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        550..625
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        724..799
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        898..973
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1284..1304)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1284..1302)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1323..1339)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1323..1339)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1336..1358)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1347..1363
                     /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(1371..1377,1378..1395,1396..1401))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1416..1437
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1554..1571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1725..2313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1805..1824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2484..3275,3276..3344))
                     /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     3107..3126
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3345..3449)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3476..3932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3563..3582)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3773..3794
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4058..4080
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4072..4089
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4073..4089
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4099..4118
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4099..4117
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(4309..4426,4503..4596,4688..5294,5349..5482,
                     5543..5703,5777..5802))
                     /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    6117..6186
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    6428..6813
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
ORIGIN
        1 gatctgaatt cttttttgct cacctgtgat tgctcctact caaatacaaa aacatcaaat
       61 tttctgtcaa taaagcatat ttatttatat ttattttaca ggaaagaatt ccttttaaag
      121 tgtattttaa cctataatga aaaacgatta aaaaaaatac ataaaataat tcgaaaattt
      181 ttgaatagcc caggttgata aaaattcatt tcatacgttt tataacttat gcccctaagt
      241 attttttgac catagtgttt caattctaca ttaattttac agagtagaat gaaacgccac
      301 ctactcagcc aagaggcgaa aaggttagct cgccaagcag agagggcgcc agtgctcact
      361 actttttata attctcaact tctttttcca gactcagttc gtatatatag acctattttc
      421 aatttaacgt cggggctttg agtgtgtgta gacatcaagc atcggtggtt cagtggtaga
      481 atgctcgcct gccacgcggg cggcccgggt tcgattcccg gccgatgcag ttacacagcc
      541 cttggctggg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      601 tgaaaaagtg gcaccgagtc ggtgctaaca aagcaccagt ggtctagtgg tagaatagta
      661 ccctgccacg gtacagaccc gggttcgatt cccggctggt gcactgtgct acccaatgtg
      721 tgtgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      781 agtggcaccg agtcggtgct aacaaagcac cagtggtcta gtggtagaat agtaccctgc
      841 cacggtacag acccgggttc gattcccggc tggtgcaaca gaaaagcatc ttacggagtt
      901 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      961 accgagtcgg tgcttttttg cctacctgga gcctgagagt tgttcaataa aataaaaatg
     1021 tttcgttttt ttgctttcgc cagtatttat tatttttcat caatatgtat tcaatttggt
     1081 atgtatttag taattgtaat atatagacaa tggttttccg ttgacgtaca tacatctgac
     1141 gtgtgtttat ttagacataa tagttatgtt ttcacatctt tttaatgttc gcttaatgcg
     1201 tatgcattct agacaattgt gctcggcaac agtatatttg tggtgtgcca accaacaacc
     1261 tgcaggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     1321 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1381 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1441 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1501 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     1561 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1621 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1681 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1741 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1801 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1861 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     1921 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1981 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2041 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2101 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2161 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2221 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2281 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2341 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2401 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2461 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2521 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2581 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     2641 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2701 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     2761 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2821 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     2881 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     2941 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3001 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3061 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3121 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3181 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3241 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3301 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3361 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3421 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg
     3481 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3541 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3601 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3661 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3721 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3781 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3841 attttgccga tttcggccta ttggttaaaa aaatgagctg atttaacaaa aatttaacgc
     3901 gaattttaac aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt
     3961 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     4021 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     4081 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac aagcttggat
     4141 ttattttgtt atgttatatg tattatatgt cagacataaa gaaaaggaac acatcaaatg
     4201 tgataacaaa gactaaacaa gtaattttat tacaccaaaa cgacaaaaca gtaggcagaa
     4261 caaacaacgc atagccaaac attgacgaat tggataccct gccgattgtc agacactttt
     4321 gttgatcagt ttcttgcgaa tggtctcgtc cagcggtgga atcgcctcgc ggggaatcag
     4381 aaaagtggac agattgaaca gatccagaaa caccttgtac cgatcactga aaccaaaaaa
     4441 aaacaaaggg agaacagttt gagttcattg atccccgata taatcacatc tgcgatgatc
     4501 acctgagagt ggagcgcaga tattgatatc cagacgagcc accagtgccc aactgttggg
     4561 atccaatcat gcgttgcacc atgatcacgt gattgtctgc ggcgggaata gaaagtattt
     4621 ggttaggaaa accagtctta aacataagat atatttataa aagagtatca aagaatgcaa
     4681 tacttacatc tccacttggt tattaacgag tcgatgtcca tgagcagggt gagcaactgg
     4741 tgtggttggc tgaacctggg ttcatcccta tagaaggtga tcatgatggc tccctgaagg
     4801 gcacgatggc taaaccggcg atccccacga cgcaccagtg catcgtgcac tgccggatca
     4861 aagatggagc gatacacctc gcgtcgcttc tcaatgtcca tgaggcggta gtttttcgcc
     4921 ttctccacgg gctcctccat ggcgctctgt acctgcgcct ccaggaatcg atcgacgctc
     4981 tcctgaaact tggcccagaa gttgaagcca ctctcctcca gtccgggcgt cctctccagc
     5041 catcgctgca ctagctccag tagcgaggga tctttctccg agttgcgaat cgagttccgc
     5101 gcctcctcgt cgctaaagac atccgagtac ttctggttgt atctcacccg ctgctctgtc
     5161 agaactccca gcttgttctc gatcaaacgg aactgcagcg actgaaaacc agatgcgggt
     5221 gccaggtact tgcggaagtc catgaagtct agcggggtca tggtctccag aatgggcact
     5281 tggtccacca ggagctgtac aaaggaagtt ataaacggat tttggtaaga gattcagaaa
     5341 gcactcactt ttagaatcag aaccactcgg ttcagtcgct tgacaatctc cagcgtcttg
     5401 gtttcatcga tgacctctgc atccaacatg tctcgtatgg agtcgaactc aaagatgatc
     5461 tgcttgaacc aaagctcgta ggctgtggcg aaggtactta aatgccattg agtgttgtca
     5521 tcaaagttgt aaacctactc accctggtgc gtgatgatga acagatgctc atcgtgcacg
     5581 ggtcgcttgt cctcctcgga cagcatacac tgggcatcca gcagtttgtc cagcatcaga
     5641 tactctccat agattttgcc cacttccgtg gttaatggca ccgccgaatc atcgtgatcg
     5701 tttctgtatg ggtttgaatt gaatcgcaga actgaagatc gattggcatt cctggacagc
     5761 acgtgctggt gctcacccgt ttcctgcata gggacagctc atggtgcaca gctcagatca
     5821 gatcgtgact cctcgagcgg cggatgctgg cgaactgatc tccgccagcg gaccggagat
     5881 gagaccccag cgaaccgata acagagcgag agagctccag ttccgactga ttgcacagtc
     5941 ggtgatctgg gcgatgggca ctgccagata ggctgggaat tatcaatcac ttgaggtgaa
     6001 agtgcggcgc acacaaatcc aagcttgata tcatcgatct cgaggctgca tccaacgcgt
     6061 tgggagctct ccggatcaat tcggcttcag gtaccgtcga cgatgtaggt cacggtctcg
     6121 aagccgcggt gcgggtgcca gggcgtgccc ttgggctccc cgggcgcgta ctccacctca
     6181 cccatctggt ccatcatgat gaacgggtcg aggtggcggt agttgatccc ggcgaacgcg
     6241 cggcgcaccg ggaagccctc gccctcgaaa ccgctgggcg cggtggtcac ggtgagcacg
     6301 ggacgtgcga cggcgtcggc tggtgcggat acgcggggca gcgtcagcgg gttctcgacg
     6361 gtcacggcgg gcatgtcgac aagccgaatt gatccactag aaggcctaat tcggtacact
     6421 agttggccac gtaataagtg tgcgttgaat ttattcgcaa aaacattgca tattttcggc
     6481 aaagtaaaat tttgttgcat accttatcaa aaaataagtg ctgcatactt tttagagaaa
     6541 ccaaataatt ttttattgca tacccgtttt taataaaata cattgcatac cctcttttaa
     6601 taaaaaatat tgcatacttt gacgaaacaa attttcgttg catacccaat aaaagattat
     6661 tatattgcat acccgttttt aataaaatac attgcatacc ctcttttaat aaagaatatt
     6721 gcatacgttg acgaaacaaa ttttcgttgc atacccaata aaagattatt atattgcata
     6781 ccttttcttg ccataccatt tagccgatca attga
//
