LOCUS       sequence-261619-        6238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone vector for CRISPR-HDR insertion of SPARC-Variant constructs
            (or any module) near the attP40 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6238)
  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 6238)
  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: pHD-3XP3-dsRed-DattP-CRISPR-donor-attP40
FEATURES             Location/Qualifiers
     source          1..6238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1163..1196
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1422..1431
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1428..2105
                     /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
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(1558..1576)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1608..1628)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1826..1845
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2020..2043
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2226..2347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2263..2282)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2317..2336
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2354..2387
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4046..4063)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4046..4062)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4161..4178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4332..4920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4412..4431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5042..5833,5834..5902))
                     /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     5665..5684
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5903..5994)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6036..6055
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6061..6080
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tttcgcccgg gctaattatg gggtgtcgcc cttcgcacag aaaagcatct tacggatggt
       61 gtttgccaaa agtgctggag atgttttaga tatagatggt ttattttaag aaatggatgt
      121 atataattga gctactgcat cacgggtttc tgtttaacag taacagtgaa aagtgctcaa
      181 tgatacatat attacaaata ataacttttg gtttttaaag tagatttata taatataatc
      241 ataattctta atgggaatcc atgttacgac caaattagcc gtggcctttc aagtggctgc
      301 cacttatatt ttacaactcg cacacagtgg ctcacaccct cgtaaacaaa ataatgctat
      361 taaatcaaat tgaacatgtg aattctctca agaagacgcc actttttttt ggggggccag
      421 ctgcaactgc caccaatgtt tacttttatc tttaatgggc acatgcctta catatacgaa
      481 ttttacacga tttttcaagt ggcggtcgag agtggggtga taattaatta aggttgcctt
      541 tagataaagt attatagagt ttcaaaaaaa tgaaacttaa gaatggaata gctgcattcc
      601 attctatgta gatttccctt caaaattatt aaaatccgtt gacctttgtt catttagcaa
      661 ttagaattcg tctatgaata acaaattaaa ttgcgttgcg ggcaaattgc tttaggccat
      721 ttgaagtgca actttatatc tcagggccaa cttctcggcg cgacaacaat aaaaacatat
      781 acatagatac acatatcaga tgcaatgcaa tcaccacagc gtgacgtcat tagtctaggc
      841 ttgaaaatac acggcaaact tgtttataac tctacgctga ttacataagc cgcaaacacg
      901 acgaaaaaac atttgttttg gtttcaaatc catgaatgac tttcattaag tgcagctgta
      961 agctgagctt tgccatgcgc tgacccatat tttcagcctc tcgttctctc tgtttcgcag
     1021 atactgtgaa tattagtaaa acgatccata ggggctgtag tttataatca taatcaaata
     1081 ggcataaaca accaccagcg cggccgcgga catatgcaca cctgcgatcg acgcgtagtc
     1141 gatcgcggta ccgggggcgt agataacttc gtataatgta tgctatacga agttatcgta
     1201 cgggatctaa ttcaattaga gactaattca attagagcta attcaattag gatccaagct
     1261 tatcgatttc gaaccctcga ccgccggagt ataaatagag gcgcttcgtc tacggagcga
     1321 caattcaatt caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca
     1381 agcgcagctg aacaagctaa acaatcggct cgaagccggt cgccaccatg gcctcctccg
     1441 aggacgtcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc
     1501 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     1561 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt
     1621 tccagtacgg ctccaaggtg tacgtgaagc accccgccga catccccgac tacaagaagc
     1681 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     1741 tgaccgtgac ccaggactcc tccctccagg acggctcctt catctacaag gtgaagttca
     1801 tcggcgtgaa cttcccctcc gacggccccg taatgcagaa gaagactatg ggctgggagg
     1861 cgtccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc cacaaggccc
     1921 tgaagctgaa ggacggcggc cactacctgg tggagttcaa gtccatctac atggccaaga
     1981 agcccgtgca gctgcccggc tactactacg tggactccaa gctggacatc acctcccaca
     2041 acgaggacta caccatcgtg gagcagtacg agcgcgccga gggccgccac cacctgttcc
     2101 tgtaggggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     2161 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     2221 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2281 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2341 tatcttaacc ggtataactt cgtataatgt atgctatacg aagttatcac attgggtagc
     2401 acagactctg agctttacta cgattactat acagctcttc tctcgcgact ttttggactg
     2461 gacaaggcgt agcacattga acggcagtgg gtttgggttt agtatcgaac cggctttcta
     2521 cgacagcgga ttggaagcgc ggagcgacaa agtcgccggg taataatgtg atatagccgg
     2581 ctatgttcag caggcaaaac tgaaataaaa gtaataaaca ccgaaagccc ccgaattctc
     2641 gcatcttctt ttgtgggact gcgagaagaa ttgcgagcga aatactaatt tccttaccta
     2701 actgtgtgag ataatctccg gttggcattg tgtgtcgcgc aataaaatcc ccaaaagccc
     2761 aaagcttcaa gtgtttaata taataaaata gccaagaagc gtgtgtattt gcatttggga
     2821 aaaatttaag gttgaattac gtgggccgaa aaattaaaga ctgaaggcca ttcaagggtt
     2881 gttcaaagag aaaattcggt gaaaagtcac cgtgtgtgtg tgtgtgagtg tgtgtgcgtt
     2941 gcgtgagaaa aatcaattct gcatttggtg gaaagccgaa aacggagaga aaattggtgg
     3001 agcgaggaga aaaggacacg aattaaaggc tggttttttg gggctgcgaa aaactcgtga
     3061 ccgatgacaa tttggcttga cgagggtcta ttagcgaaat caatgctatt atggctgata
     3121 gtggaggacc aggatccaag cacatggatc caacaaccat agatgccggc gggggcggag
     3181 caggagcagc cggaggtgat gacgtgcctc cagtcccagc tgttcgccgg cgtcgtgcgc
     3241 atgagcagaa atcctcgaga gagcaggtgc tggaggagga gaagagccaa cagctggaga
     3301 catcgacgat gacgaggacc tacatgaaga ccatcaccac ctccctcacc acctcgagta
     3361 gctccaacgt ggaagagttt atcctggggg agcacgcagc aggagcagca gctgctccat
     3421 cgcccaacca gcagcgattg cgtcaaaagg tggcccagta cgagaaggtc tggtcggatg
     3481 gctccagtcc cgttaagcgt ccagccgagc agagctctga tgagctgcgt ctgaccgacg
     3541 accaggatga gtacgccatc cgtaagatgc ttttctgtgt caatcgagtt caagggcgac
     3601 acaaaattta ttctaaatgc ataataaata ctgataacat cttatagttt gtattatatt
     3661 ttgtattatc gttgacatgt ataattttga tatcaaaaac tgattttccc tttattattt
     3721 tcgagattta ttttcttaat tctctttaac aaactagaaa tattgtatat acaaaaaatc
     3781 ataaataata gatgaatagt ttaattatag gtgttcatca atcgaaaaag caacgtatct
     3841 tatttaaagt gcgttgcttt tttctcattt ataaggttaa ataattctca tatatcaagc
     3901 aaagtgacag gcgcccttaa atattctgac aaatgctctt tccctaaact ccccccataa
     3961 aaaaacccgc cgaagcgggt ttttacgtta tttgcggatt aacgattact cgttatcaga
     4021 accgcccagg gggcccgagc ttaagactgg ccgtcgtttt acaacacaga aagagtttgt
     4081 agaaacgcaa aaaggccatc cgtcaggggc cttctgctta gtttgatgcc tggcagttcc
     4141 ctactctcgc cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4201 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4261 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4321 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4381 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4441 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4501 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4561 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4621 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4681 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4741 aagtggtggg ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4801 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4861 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4921 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgacgc gcgcgtaact
     4981 cacgttaagg gattttggtc atgagcttgc gccgtcccgt caagtcagcg taatgctctg
     5041 cttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5101 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5161 ccagcgctgc gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa
     5221 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5281 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5341 acgttgttgc catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5401 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5461 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5521 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5581 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5641 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5701 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5761 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5821 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5881 cacggaaatg ttgaatactc atattcttcc tttttcaata ttattgaagc atttatcagg
     5941 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6001 tcagtgttac aaccaattaa ccaattctga acattatcgc gagcccattt atacctgaat
     6061 atggctcata acaccccttg tttgcctggc ggcagtagcg cggtggtccc acctgacccc
     6121 atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggac tccccatgcg
     6181 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcc
//
