LOCUS       sequence-256619-        4081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone vector for CRISPR-HDR insertion of SPARC-Variant 
            constructs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4081)
  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 4081)
  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
FEATURES             Location/Qualifiers
     source          1..4081
                     /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    1322..1355
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1581..1590
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1587..2264
                     /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(1717..1735)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1767..1787)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1985..2004
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2179..2202
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    2385..2506
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2422..2441)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2476..2495
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2513..2546
                     /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(3082..3099)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3082..3098)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3197..3214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3368..3956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3448..3467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4080..4081,1..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 ctgaagcagg tggaattctt gcatgctagc
     1261 ggccgcggac atatgcacac ctgcgatcga cgcgtagtcg atcgcggtac cgggggcgta
     1321 gataacttcg tataatgtat gctatacgaa gttatcgtac gggatctaat tcaattagag
     1381 actaattcaa ttagagctaa ttcaattagg atccaagctt atcgatttcg aaccctcgac
     1441 cgccggagta taaatagagg cgcttcgtct acggagcgac aattcaattc aaacaagcaa
     1501 agtgaacacg tcgctaagcg aaagctaagc aaataaacaa gcgcagctga acaagctaaa
     1561 caatcggctc gaagccggtc gccaccatgg cctcctccga ggacgtcatc aaggagttca
     1621 tgcgcttcaa ggtgcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1681 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg
     1741 gccccctgcc cttcgcctgg gacatcctgt ccccccagtt ccagtacggc tccaaggtgt
     1801 acgtgaagca ccccgccgac atccccgact acaagaagct gtccttcccc gagggcttca
     1861 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     1921 ccctccagga cggctccttc atctacaagg tgaagttcat cggcgtgaac ttcccctccg
     1981 acggccccgt aatgcagaag aagactatgg gctgggaggc gtccaccgag cgcctgtacc
     2041 cccgcgacgg cgtgctgaag ggcgagatcc acaaggccct gaagctgaag gacggcggcc
     2101 actacctggt ggagttcaag tccatctaca tggccaagaa gcccgtgcag ctgcccggct
     2161 actactacgt ggactccaag ctggacatca cctcccacaa cgaggactac accatcgtgg
     2221 agcagtacga gcgcgccgag ggccgccacc acctgttcct gtaggggccg cgactctaga
     2281 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     2341 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     2401 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2461 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaccg gtataacttc
     2521 gtataatgta tgctatacga agttatagaa gagcactagt aaagatctcc atgcataagg
     2581 cgcgcctagg ccttctgcag ctcgaggctc ttccgtcaat cgagttcaag ggcgacacaa
     2641 aatttattct aaatgcataa taaatactga taacatctta tagtttgtat tatattttgt
     2701 attatcgttg acatgtataa ttttgatatc aaaaactgat tttcccttta ttattttcga
     2761 gatttatttt cttaattctc tttaacaaac tagaaatatt gtatatacaa aaaatcataa
     2821 ataatagatg aatagtttaa ttataggtgt tcatcaatcg aaaaagcaac gtatcttatt
     2881 taaagtgcgt tgcttttttc tcatttataa ggttaaataa ttctcatata tcaagcaaag
     2941 tgacaggcgc ccttaaatat tctgacaaat gctctttccc taaactcccc ccataaaaaa
     3001 acccgccgaa gcgggttttt acgttatttg cggattaacg attactcgtt atcagaaccg
     3061 cccagggggc ccgagcttaa gactggccgt cgttttacaa cacagaaaga gtttgtagaa
     3121 acgcaaaaag gccatccgtc aggggccttc tgcttagttt gatgcctggc agttccctac
     3181 tctcgccttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3241 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3301 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3361 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3421 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3481 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3541 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3601 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3661 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3721 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3781 ggtgggctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3841 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3901 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3961 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgacgcgcgc gtaactcacg
     4021 ttaagggatt ttggtcatga gcttgcgccg tcccgtcaag tcagcgtaat gctctgcttt
     4081 t
//
