LOCUS       sequence-178932-        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"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1078..1134
                     /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             1135..1845
                     /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(1280..1298)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1548..1567
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2707..2726)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2708..2726)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2733..2750)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2733..2749)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2742..2764)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2890..3318
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2977..2996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3187..3208
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3662..4456
                     /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(3716..3735)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4326..4345
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4474..4542,4543..5334)
                     /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(4692..4711)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5505..6093
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5994..6013
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6247..6264
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtacctgccc ctgtcgcaca tgtgaggggg tcccgggggc agcggcgtaa gatggcccag
      301 gagaacccca agatgcacaa ctcggagatc agcaagcgcc tgggcgcgga gtggaaactt
      361 ttgtccgaga ccgagaagcg gccgttcatc gacgaggcca agcggctgcg cgctctgcac
      421 atgaaggagc acccggatta taaataccgg ccgcggcgga aaaccaagac gctcatgaag
      481 aaggataagt acacgcttcc cggaggcttg ctggcccccg gcgggaacag catggcgagc
      541 ggggttgggg tgggcgccgg cctgggtgcg ggcgtgaacc agcgcatgga cagctacgcg
      601 cacatgaacg gctggagcaa cggcagctac agcatgatgc aggagcagct gggctacccg
      661 cagcacccgg gcctcaacgc tcacggcgcg gcacagatgc aaccgatgca ccgctacgac
      721 gtcagcgccc tgcagtacaa ctccatgacc agctcgcaga cctacatgaa cggctcgccc
      781 acctacagca tgtcctactc gcagcagggc acccccggta tggcgctggg ctccatgggc
      841 tctgtggtca agtccgaggc cagctccagc ccccccgtgg ttacctcttc ctcccactcc
      901 agggcgccct gccaggccgg ggacctccgg gacatgatca gcatgtacct ccccggcgcc
      961 gaggtgccgg agcccgctgc gcccagtaga ctgcacatgg cccagcacta ccagagcggc
     1021 ccggtgcccg gcacggccat taacggcaca ctgcccctgt cgcacatgtt aattaacgcc
     1081 actaacttct ccctgttgaa acaagcaggg gatgtcgaag agaatcccgg gccaatggtg
     1141 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     1201 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     1261 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     1321 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     1381 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     1441 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     1501 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     1561 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     1621 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     1681 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     1741 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     1801 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaggcgc gcctgagggc
     1861 tggactgcga actggagaag gggagagatt ttcaaagaga tacaagggaa ttgggagggg
     1921 tgcaaaaaga ggagagtagg aaaaatctga taatgctcaa aaggaaaaaa aatctccgca
     1981 gcgaaacgac agctgcggaa aaaaaccacc aatcccatcc aaattaacgc aaaaaccgtg
     2041 atgccgacta gaaaactttt atgagagatc ttgggacttc tttttggggg actatttttg
     2101 tacagagaaa acctgagggc ggcggggagg gcgggggaat cggaccatgt atagatctgg
     2161 aggaaaaaaa ctacgcaaaa ctttttttta aagttctagt ggtacgttag gcgcttcgca
     2221 gggagttcgc aaaagtcttt accagtaata tttagagcta gactccgggc gatgaaaaaa
     2281 aagttttaat atttgcaagc aacttttgta cagtatttat cgagataaac atggcaatca
     2341 aatgtccatt gtttataagc tgagaatttg ccaatatttt tcgaggaaag ggttcttgct
     2401 gggttttgat tctgcagctt aaatttagga ccgttacaaa caaggaagga gtttattcgg
     2461 atttgaacat tttagtttta aaattgtaca aaaggaaaac atgagagcaa gtactggcaa
     2521 gaccgttttc gtggtcttgt ttaaggcaaa cgttctagat tgtactaaat ttttaactta
     2581 ctgttaaagg caaaaaaaaa atgtccatgc aggttgatat cgttggtaat ttataatagc
     2641 ttttgttcaa tccccctcac atgtgcgaca ggggcactcg agcatgcatc tagagggccc
     2701 aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac
     2761 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2821 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2881 ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2941 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3001 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3061 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3121 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3181 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3241 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3301 aaaaatttaa cgcgaatttt aacaaaattc agggcgcaag ggctgctaaa ggaagcggaa
     3361 cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc
     3421 tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac
     3481 atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg
     3541 ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc
     3601 aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg
     3661 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3721 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3781 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3841 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3901 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3961 ggatctcctg tcatcccacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4021 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4081 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4141 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4201 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4261 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4321 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4381 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4441 tgacgagttc ttctgaattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4501 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4561 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4621 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4681 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     4741 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4801 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4861 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4921 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4981 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5041 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5101 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5161 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5221 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5281 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5341 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5401 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5461 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5521 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5581 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5641 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5701 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5761 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5821 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5881 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5941 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6001 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6061 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6121 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6181 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     6241 gagcgcagcg agtcagtgag cgaggaagcg gaag
//
