LOCUS       sequence-182791-        6274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HMEJ donor for fusing a p2A-mCherry reporter to mouse Sox2. AAV 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6274)
  AUTHORS   Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, 
            Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang
            P, Sun Q, Shi L, Yang H
  TITLE     Homology-mediated end joining-based targeted integration using 
            CRISPR/Cas9.
  JOURNAL   Cell Res. 2017 May 19. doi: 10.1038/cr.2017.76.
  PUBMED    28524166
REFERENCE   2  (bases 1 to 6274)
  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"; journalName: "Cell Res. 
            2017 May 19. doi: 10.1038/cr.2017.76."
COMMENT     Sequence Label: AAV_Sox2 HMEJ donor
FEATURES             Location/Qualifiers
     source          1..6274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     163..182
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1322..1341)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1323..1341)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1348..1365)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1348..1364)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1357..1379)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1505..1933
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1592..1611)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1802..1823
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             2277..3071
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2331..2350)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2941..2960
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3089..3157,3158..3949)
                     /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(3307..3326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4120..4708
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4609..4628
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4862..4879
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4996..5017
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5032..5037,5038..5055,5056..5062)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5070..5086
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5075..5097
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5094..5110
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5094..5110
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5967..6023
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(6024..6274,1..460)
                     /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(6169..6187)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
ORIGIN
        1 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
       61 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
      121 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
      181 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
      241 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
      301 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
      361 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
      421 ccgccactcc accggcggca tggacgagct gtacaagtaa ggcgcgcctg agggctggac
      481 tgcgaactgg agaaggggag agattttcaa agagatacaa gggaattggg aggggtgcaa
      541 aaagaggaga gtaggaaaaa tctgataatg ctcaaaagga aaaaaaatct ccgcagcgaa
      601 acgacagctg cggaaaaaaa ccaccaatcc catccaaatt aacgcaaaaa ccgtgatgcc
      661 gactagaaaa cttttatgag agatcttggg acttcttttt gggggactat ttttgtacag
      721 agaaaacctg agggcggcgg ggagggcggg ggaatcggac catgtataga tctggaggaa
      781 aaaaactacg caaaactttt ttttaaagtt ctagtggtac gttaggcgct tcgcagggag
      841 ttcgcaaaag tctttaccag taatatttag agctagactc cgggcgatga aaaaaaagtt
      901 ttaatatttg caagcaactt ttgtacagta tttatcgaga taaacatggc aatcaaatgt
      961 ccattgttta taagctgaga atttgccaat atttttcgag gaaagggttc ttgctgggtt
     1021 ttgattctgc agcttaaatt taggaccgtt acaaacaagg aaggagttta ttcggatttg
     1081 aacattttag ttttaaaatt gtacaaaagg aaaacatgag agcaagtact ggcaagaccg
     1141 ttttcgtggt cttgtttaag gcaaacgttc tagattgtac taaattttta acttactgtt
     1201 aaaggcaaaa aaaaaatgtc catgcaggtt gatatcgttg gtaatttata atagcttttg
     1261 ttcaatcccc ctcacatgtg cgacaggggc actcgagcat gcatctagag ggcccaattc
     1321 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
     1381 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     1441 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     1501 atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1561 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1621 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1681 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1741 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1801 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1861 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1921 tttaacgcga attttaacaa aattcagggc gcaagggctg ctaaaggaag cggaacacgt
     1981 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct
     2041 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc
     2101 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc
     2161 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga
     2221 tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga
     2281 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2341 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     2401 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     2461 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     2521 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     2581 tcctgtcatc ccaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     2641 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     2701 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     2761 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     2821 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     2881 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     2941 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3001 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3061 agttcttctg aattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3121 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3181 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3241 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3301 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3361 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3421 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3481 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3541 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3601 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3661 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3721 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3781 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3841 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3901 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3961 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4021 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4081 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4141 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4201 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4261 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4321 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4381 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4441 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4501 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4561 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4621 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4681 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4741 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4801 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4861 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     4921 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4981 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     5041 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     5101 cagctatgac catgattacg ccaagcttgg tacctgcccc tgtcgcacat gtgagggggt
     5161 cccgggggca gcggcgtaag atggcccagg agaaccccaa gatgcacaac tcggagatca
     5221 gcaagcgcct gggcgcggag tggaaacttt tgtccgagac cgagaagcgg ccgttcatcg
     5281 acgaggccaa gcggctgcgc gctctgcaca tgaaggagca cccggattat aaataccggc
     5341 cgcggcggaa aaccaagacg ctcatgaaga aggataagta cacgcttccc ggaggcttgc
     5401 tggcccccgg cgggaacagc atggcgagcg gggttggggt gggcgccggc ctgggtgcgg
     5461 gcgtgaacca gcgcatggac agctacgcgc acatgaacgg ctggagcaac ggcagctaca
     5521 gcatgatgca ggagcagctg ggctacccgc agcacccggg cctcaacgct cacggcgcgg
     5581 cacagatgca accgatgcac cgctacgacg tcagcgccct gcagtacaac tccatgacca
     5641 gctcgcagac ctacatgaac ggctcgccca cctacagcat gtcctactcg cagcagggca
     5701 cccccggtat ggcgctgggc tccatgggct ctgtggtcaa gtccgaggcc agctccagcc
     5761 cccccgtggt tacctcttcc tcccactcca gggcgccctg ccaggccggg gacctccggg
     5821 acatgatcag catgtacctc cccggcgccg aggtgccgga gcccgctgcg cccagtagac
     5881 tgcacatggc ccagcactac cagagcggcc cggtgcccgg cacggccatt aacggcacac
     5941 tgcccctgtc gcacatgtta attaacgcca ctaacttctc cctgttgaaa caagcagggg
     6001 atgtcgaaga gaatcccggg ccaatggtga gcaagggcga ggaggataac atggccatca
     6061 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
     6121 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     6181 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
     6241 gctccaaggc ctacgtgaag caccccgccg acat
//
