LOCUS       sequence-261127-        4079 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 4079)
  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 4079)
  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..4079
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    133..166
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      392..401
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             398..1075
                     /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(528..546)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(578..598)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     796..815
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     990..1013
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1196..1317
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1233..1252)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1287..1306
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1324..1357
                     /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(1893..1910)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1893..1909)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2008..2025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2179..2767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2259..2278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2889..3680,3681..3749))
                     /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     3512..3531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3750..3841)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3883..3902
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3908..3927
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tgggcctttc gcccgggcta attatggggt gtcgcccttc gctgaagcag gtggaattct
       61 tgcatgctag cggccgcgga catatgcaca cctgcgatcg acgcgtagtc gatcgcggta
      121 ccgggggcgt agataacttc gtataatgta tgctatacga agttatcgta cgggatctaa
      181 ttcaattaga gactaattca attagagcta attcaattag gatccaagct tatcgatttc
      241 gaaccctcga ccgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt
      301 caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg
      361 aacaagctaa acaatcggct cgaagccggt cgccaccatg gcctcctccg aggacgtcat
      421 caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc acgagttcga
      481 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
      541 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt tccagtacgg
      601 ctccaaggtg tacgtgaagc accccgccga catccccgac tacaagaagc tgtccttccc
      661 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
      721 ccaggactcc tccctccagg acggctcctt catctacaag gtgaagttca tcggcgtgaa
      781 cttcccctcc gacggccccg taatgcagaa gaagactatg ggctgggagg cgtccaccga
      841 gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc cacaaggccc tgaagctgaa
      901 ggacggcggc cactacctgg tggagttcaa gtccatctac atggccaaga agcccgtgca
      961 gctgcccggc tactactacg tggactccaa gctggacatc acctcccaca acgaggacta
     1021 caccatcgtg gagcagtacg agcgcgccga gggccgccac cacctgttcc tgtaggggcc
     1081 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     1141 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     1201 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     1261 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacc
     1321 ggtataactt cgtataatgt atgctatacg aagttataga agagcactag taaagatctc
     1381 catgcataag gcgcgcctag gccttctgca gctcgaggct cttccgtcaa tcgagttcaa
     1441 gggcgacaca aaatttattc taaatgcata ataaatactg ataacatctt atagtttgta
     1501 ttatattttg tattatcgtt gacatgtata attttgatat caaaaactga ttttcccttt
     1561 attattttcg agatttattt tcttaattct ctttaacaaa ctagaaatat tgtatataca
     1621 aaaaatcata aataatagat gaatagttta attataggtg ttcatcaatc gaaaaagcaa
     1681 cgtatcttat ttaaagtgcg ttgctttttt ctcatttata aggttaaata attctcatat
     1741 atcaagcaaa gtgacaggcg cccttaaata ttctgacaaa tgctctttcc ctaaactccc
     1801 cccataaaaa aacccgccga agcgggtttt tacgttattt gcggattaac gattactcgt
     1861 tatcagaacc gcccaggggg cccgagctta agactggccg tcgttttaca acacagaaag
     1921 agtttgtaga aacgcaaaaa ggccatccgt caggggcctt ctgcttagtt tgatgcctgg
     1981 cagttcccta ctctcgcctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2041 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2101 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2161 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2221 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2281 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2341 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2401 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2461 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2521 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2581 gttcttgaag tggtgggcta actacggcta cactagaaga acagtatttg gtatctgcgc
     2641 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2701 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2761 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgacgcgcg
     2821 cgtaactcac gttaagggat tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa
     2881 tgctctgctt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2941 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3001 tctggcccca gcgctgcgat gataccgcga gaaccacgct caccggctcc ggatttatca
     3061 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3121 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3181 ttgcgcaacg ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3241 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3301 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3361 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3421 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3481 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3541 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3601 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3661 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3721 agggcgacac ggaaatgttg aatactcata ttcttccttt ttcaatatta ttgaagcatt
     3781 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3841 ataggggtca gtgttacaac caattaacca attctgaaca ttatcgcgag cccatttata
     3901 cctgaatatg gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc
     3961 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc
     4021 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagac
//
