LOCUS       sequence-394120-        6093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mCherry in a Cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6093)
  AUTHORS   Moffa JC, Bland IN, Tooley JR, Kalyanaraman V, Heitmeier M, Creed 
            MC, Copits BA
  TITLE     Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and 
            Genetically Encoded Tool Delivery in the Central and Peripheral 
            Nervous Systems.
  JOURNAL   eNeuro. 2024 Jul 5;11(7):ENEURO.0438-23.2024. doi: 
            10.1523/ENEURO.0438-23.2024. Print 2024 Jul.
  PUBMED    38871457
REFERENCE   2  (bases 1 to 6093)
  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: 
            "10.1523/ENEURO.0438-23.2024"; journalName: "eNeuro"; date: 
            "2024-07-5- 5"; volume: "11"; issue: "7"; pages: 
            "ENEURO.0438-23.2024"
COMMENT     Sequence Label: pX552-EF1a-DIO-mcherry(with gRNA scaffold)
FEATURES             Location/Qualifiers
     source          1..6093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        459..1637
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          690..1628
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1585..1605
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1668..1701)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1752..1785)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1797..2507)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2075..2094)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2344..2362
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    2529..2562
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2613..2646
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2696..3172
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2895..2914)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3212..3352
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3212..3341
                     /label=AAV2 ITR
     rep_origin      3427..3882
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3514..3533)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3724..3745
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3899..3918)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4018..4040
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4078..4096)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4164..4268
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4269..4337,4338..5129)
                     /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     complement(4487..4506)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5300..5888
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5789..5808
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5950..6079
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gaagagcgag ctcttctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagactgca
      361 gagggccatc gatagctttg caaagatgga taaagtttta aacagagagg aatctttgca
      421 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
      481 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
      541 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
      601 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
      661 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
      721 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
      781 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
      841 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
      901 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
      961 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     1021 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     1081 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     1141 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     1201 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     1261 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     1321 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     1381 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     1441 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     1501 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     1561 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     1621 catttcaggt gtcgtgagga attggatcct ctagagtcga ctccggaata acttcgtata
     1681 ggatacttta tacgaagtta tgcagaatgg tagctggatt gtagctgcta ttagcaatat
     1741 gaaacctctt aataacttcg tatagcatac attatacgaa gttatggcgc gccttattac
     1801 ttgtacagct cgtccatgcc gccggtggag tggcggccct cggcgcgttc gtactgttcc
     1861 acgatggtgt agtcctcgtt gtgggaggtg atgtccaact tgatgttgac gttgtaggcg
     1921 ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcagc gtcgtagtgg
     1981 ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg
     2041 tacatccgct cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg
     2101 gaggggaagt tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag
     2161 gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg
     2221 aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag
     2281 gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggcca
     2341 cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg
     2401 ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg
     2461 aactccttga tgatggccat gttatcctcc tcgcccttgc tcaccattgg cccggggcta
     2521 gcgctagcat aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat
     2581 tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga
     2641 agttatgaat tcgatatcaa gcttatcgat accgagcgct gctcgagaga tctacgggtg
     2701 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     2761 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     2821 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     2881 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     2941 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     3001 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     3061 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     3121 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     3181 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     3241 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     3301 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     3361 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     3421 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3481 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     3541 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3601 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     3661 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3721 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     3781 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     3841 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     3901 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     3961 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4021 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4081 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4141 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4201 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4261 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4321 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4381 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4441 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4501 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4561 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4621 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4681 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4741 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4801 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4861 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4921 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4981 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5041 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5101 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5161 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5221 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5281 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5341 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5401 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5461 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5521 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5581 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5641 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5701 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5761 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5821 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5881 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5941 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
     6001 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
     6061 tccatcacta ggggttcctg cggccgcacg cgt
//
