LOCUS       sequence-256620-        6234 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 6234)
  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 6234)
  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..6234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     622..641
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(860..951)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     993..1012
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1018..1037
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    2358..2391
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2617..2626
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2623..3300
                     /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(2753..2771)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2803..2823)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3021..3040
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3215..3238
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3421..3542
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3458..3477)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3512..3531
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    3549..3582
                     /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(5235..5252)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5235..5251)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5350..5367)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5521..6109)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5601..5620)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6233..6234,1..790,791..859))
                     /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"
ORIGIN
        1 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
       61 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
      121 gcgctgcgat gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc
      181 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
      241 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
      301 ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
      361 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
      421 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
      481 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
      541 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
      601 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
      661 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      721 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      781 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      841 ggaaatgttg aatactcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt
      901 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca
      961 gtgttacaac caattaacca attctgaaca ttatcgcgag cccatttata cctgaatatg
     1021 gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     1081 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga
     1141 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1201 cccgggctaa ttatggggtg tcgcccttcg cacagaaaag catcttacgg atggtgtttg
     1261 ccaaaagtgc tggagatgtt ttagatatag atggtttatt ttaagaaatg gatgtatata
     1321 attgagctac tgcatcacgg gtttctgttt aacagtaaca gtgaaaagtg ctcaatgata
     1381 catatattac aaataataac ttttggtttt taaagtagat ttatataata taatcataat
     1441 tcttaatggg aatccatgtt acgaccaaat tagccgtggc ctttcaagtg gctgccactt
     1501 atattttaca actcgcacac agtggctcac accctcgtaa acaaaataat gctattaaat
     1561 caaattgaac atgtgaattc tctcaagaag acgccacttt tttttggggg gccagctgca
     1621 actgccacca atgtttactt ttatctttaa tgggcacatg ccttacatat acgaatttta
     1681 cacgattttt caagtggcgg tcgagagtgg ggtgataatt aattaaggtt gcctttagat
     1741 aaagtattat agagtttcaa aaaaatgaaa cttaagaatg gaatagctgc attccattct
     1801 atgtagattt cccttcaaaa ttattaaaat ccgttgacct ttgttcattt agcaattaga
     1861 attcgtctat gaataacaaa ttaaattgcg ttgcgggcaa attgctttag gccatttgaa
     1921 gtgcaacttt atatctcagg gccaacttct cggcgcgaca acaataaaaa catatacata
     1981 gatacacata tcagatgcaa tgcaatcacc acagcgtgac gtcattagtc taggcttgaa
     2041 aatacacggc aaacttgttt ataactctac gctgattaca taagccgcaa acacgacgaa
     2101 aaaacatttg ttttggtttc aaatccatga atgactttca ttaagtgcag ctgtaagctg
     2161 agctttgcca tgcgctgacc catattttca gcctctcgtt ctctctgttt cgcagatact
     2221 gtgaatatta gtaaaacgat ccataggggc tgtagtttat aatcataatc aaataggcat
     2281 aaacaaccac cagcgcggcc gcggacatat gcacacctgc gatcgacgcg tagtcgatcg
     2341 cggtaccggg ggcgtagata acttcgtata atgtatgcta tacgaagtta tcgtacggga
     2401 tctaattcaa ttagagacta attcaattag agctaattca attaggatcc aagcttatcg
     2461 atttcgaacc ctcgaccgcc ggagtataaa tagaggcgct tcgtctacgg agcgacaatt
     2521 caattcaaac aagcaaagtg aacacgtcgc taagcgaaag ctaagcaaat aaacaagcgc
     2581 agctgaacaa gctaaacaat cggctcgaag ccggtcgcca ccatggcctc ctccgaggac
     2641 gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag
     2701 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     2761 aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttccag
     2821 tacggctcca aggtgtacgt gaagcacccc gccgacatcc ccgactacaa gaagctgtcc
     2881 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     2941 gtgacccagg actcctccct ccaggacggc tccttcatct acaaggtgaa gttcatcggc
     3001 gtgaacttcc cctccgacgg ccccgtaatg cagaagaaga ctatgggctg ggaggcgtcc
     3061 accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg agatccacaa ggccctgaag
     3121 ctgaaggacg gcggccacta cctggtggag ttcaagtcca tctacatggc caagaagccc
     3181 gtgcagctgc ccggctacta ctacgtggac tccaagctgg acatcacctc ccacaacgag
     3241 gactacacca tcgtggagca gtacgagcgc gccgagggcc gccaccacct gttcctgtag
     3301 gggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta
     3361 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     3421 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3481 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3541 taaccggtat aacttcgtat aatgtatgct atacgaagtt atcacattgg gtagcacaga
     3601 ctctgagctt tactacgatt actatacagc tcttctctcg cgactttttg gactggacaa
     3661 ggcgtagcac attgaacggc agtgggtttg ggtttagtat cgaaccggct ttctacgaca
     3721 gcggattgga agcgcggagc gacaaagtcg ccgggtaata atgtgatata gccggctatg
     3781 ttcagcaggc aaaactgaaa taaaagtaat aaacaccgaa agcccccgaa ttctcgcatc
     3841 ttcttttgtg ggactgcgag aagaattgcg agcgaaatac taatttcctt acctaactgt
     3901 gtgagataat ctccggttgg cattgtgtgt cgcgcaataa aatccccaaa agcccacaag
     3961 tgtttaatat aataaaatag ccaagaagcg tgtgtatttg catttgggaa aaatttaagg
     4021 ttgaattacg tgggccgaaa aattaaagac tgaaggccat tcaagggttg ttcaaagaga
     4081 aaattcggtg aaaagtcacc gtgtgtgtgt gtgtgagtgt gtgtgcgttg cgtgagaaaa
     4141 atcaattctg catttggtgg aaagccgaaa acggagagaa aattggtgga gcgaggagaa
     4201 aaggacacga attaaaggct ggttttttgg ggctgcgaaa aactcgtgac cgatgacaat
     4261 ttggcttgac gagggtctat tagcgaaatc aatgctatta tggctgatag tggaggacca
     4321 ggatccaagc acatggatcc aacaaccata gatgccggcg ggggcggagc aggagcagcc
     4381 ggaggtgatg acgtgcctcc agtcccagct gttcgccggc gtcgtgcgca tgagcagaaa
     4441 tcctcgagag agcaggtgct ggaggaggag aagagccaac agctggagac atcgacgatg
     4501 acgaggacct acatgaagac catcaccacc tccctcacca cctcgagtag ctccaacgtg
     4561 gaagagttta tcctggggga gcacgcagca ggagcagcag ctgctccatc gcccaaccag
     4621 cagcgattgc gtcaaaaggt ggcccagtac gagaaggtct ggtcggatgg ctccagtccc
     4681 gttaagcgtc cagccgagca gagctctgat gagctgcgtc tgaccgacga ccaggatgag
     4741 tacgccatcc gtaagatgct tttctgtgtc aatcgagttc aagggcgaca caaaatttat
     4801 tctaaatgca taataaatac tgataacatc ttatagtttg tattatattt tgtattatcg
     4861 ttgacatgta taattttgat atcaaaaact gattttccct ttattatttt cgagatttat
     4921 tttcttaatt ctctttaaca aactagaaat attgtatata caaaaaatca taaataatag
     4981 atgaatagtt taattatagg tgttcatcaa tcgaaaaagc aacgtatctt atttaaagtg
     5041 cgttgctttt ttctcattta taaggttaaa taattctcat atatcaagca aagtgacagg
     5101 cgcccttaaa tattctgaca aatgctcttt ccctaaactc cccccataaa aaaacccgcc
     5161 gaagcgggtt tttacgttat ttgcggatta acgattactc gttatcagaa ccgcccaggg
     5221 ggcccgagct taagactggc cgtcgtttta caacacagaa agagtttgta gaaacgcaaa
     5281 aaggccatcc gtcaggggcc ttctgcttag tttgatgcct ggcagttccc tactctcgcc
     5341 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5401 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5461 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5521 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5581 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5641 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5701 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5761 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5821 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5881 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtgggc
     5941 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6001 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6061 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6121 gatcttttct acggggtctg acgctcagtg gaacgacgcg cgcgtaactc acgttaaggg
     6181 attttggtca tgagcttgcg ccgtcccgtc aagtcagcgt aatgctctgc tttt
//
