LOCUS       sequence-182595-        6831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HMEJ donor for fusing a p2A-mCherry-WPRE reporter to mouse Dbh. AAV 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6831)
  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 6831)
  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_Dbh HMEJ donor
FEATURES             Location/Qualifiers
     source          1..6831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    348..936
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             454..465
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     863..883
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(937..1647)
                     /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(1215..1234)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1484..1502
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1648..1704)
                     /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"
     primer_bind     complement(2546..2562)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2546..2562)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2559..2581)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2570..2586
                     /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)."
     promoter        complement(join(2594..2600,2601..2618,2619..2624))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2639..2660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2777..2794)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2948..3536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3028..3047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3707..4498,4499..4567))
                     /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     4330..4349
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(4585..5379)
                     /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(4696..4715)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5306..5325
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(5723..6151)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5833..5854)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6045..6064
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6277..6299
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6291..6308
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6292..6308
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6315..6334
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6315..6333
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 caccaccaga ggaccaacag ggtcgggact gtggggcttg gggagtctgc acccagcctg
       61 gcaaggctgt tctggaggca aggtggtgtt gaatctggac taccacagcg agctgtccct
      121 atgtggagtc agcagtgtgt agataatgtc cagggcttct ccgttcagtt ggacctcatg
      181 gggtgcagtc atgcaagtgt ctccaaggtg acatgctcct tctccctgga tggccagcac
      241 tagtctatgg aagtgagtct tcacacagga agggagggct acacagagct ggtgtgagcc
      301 caggtggaca ggaggaggag ggtctgaaga atagcttctc acaagtggcg gggaggcggc
      361 ccaaagggag atccgactcg tctgagggcg aaggcggaga cgcggaagag gccgcagagc
      421 cggcagcagg ccgcgggaag gaaggtccgc tggattgagg gccgaaggga cgtagcagaa
      481 ggacgtcccg cgcagaatcc aggtggcaac acaggcgagc agccaaggaa aggacgatga
      541 tttccccgac aacaccacgg aattgtcagt gcccaacagc cgagcccctg tccagcagcg
      601 ggcaaggcag gcggcgatga gttccgccgt ggcaataggg agggggaaag cgaaagtccc
      661 ggaaaggagc tgacaggtgg tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac
      721 agtgcacacc acgccacgtt gcctgacaac gggccacaac tcctcataaa gagacagcaa
      781 ccaggattta tacaaggagg agaaaatgaa agccatacgg gaagcaatag catgatacaa
      841 aggcattaaa gcagcgtatc cacatagcgt aaaaggagca acatagttaa gaataccagt
      901 caatctttca caaattttgt aatccagagg ttgattttac ttgtacagct cgtccatgcc
      961 gccggtggag tggcggccct cggcgcgttc gtactgttcc acgatggtgt agtcctcgtt
     1021 gtgggaggtg atgtccaact tgatgttgac gttgtaggcg ccgggcagct gcacgggctt
     1081 cttggccttg taggtggtct tgacctcagc gtcgtagtgg ccgccgtcct tcagcttcag
     1141 cctctgcttg atctcgccct tcagggcgcc gtcctcgggg tacatccgct cggaggaggc
     1201 ctcccagccc atggtcttct tctgcattac ggggccgtcg gaggggaagt tggtgccgcg
     1261 cagcttcacc ttgtagatga actcgccgtc ctgcagggag gagtcctggg tcacggtcac
     1321 cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg ggaaggacag
     1381 cttcaagtag tcggggatgt cggcggggtg cttcacgtag gccttggagc cgtacatgaa
     1441 ctgaggggac aggatgtccc aggcgaaggg cagggggcca cccttggtca ccttcagctt
     1501 ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct cgaactcgtg
     1561 gccgttcacg gagccctcca tgtgcacctt gaagcgcatg aactccttga tgatggccat
     1621 gttatcctcc tcgcccttgc tcaccattgg cccgggattc tcttcgacat cccctgcttg
     1681 tttcaacagg gagaagttag tggcttcagc atctgcctct gttgtgatgg gcacagtggg
     1741 gttagcgggg ctctgagtct gtcggatggg gcagcgtggg gtgggttctt ccagcgtgga
     1801 ggtgatctta ggcaaaggct gcagattcca ctcaccctgt aaggaaggag cagaaggtaa
     1861 gccgaggctg gaggggccaa gcaaactgct atctccctaa ctggttggat tcttgtcctt
     1921 aaaatcattt ctcccatgct gaacactcca cacctcttcc aagtcagcct ttgagagtta
     1981 gcaccggagg atccacgtcc cttccctagg ctgttgtgtt ctctgtcagc aacctgaagc
     2041 cctccagacg tctccacctc catgcctgca ctcaactgta tactccgagg aaaggatcag
     2101 agtctttgga ggcaggccgt gggtttagcc actgtccaac aaggcgtctg caaaagccaa
     2161 gatctaacca tatccggcgg tgcctcaaac tgctctggga tgaacaagtc tccccatggc
     2221 atgggtgttt gggttagcaa ttaatccggt acccaaacgc tgccaggaaa ggtctgttca
     2281 ccttctgctg cagtgcctgc ctcccctgcc taacacagca ggcggggtct gctgaccagc
     2341 actctgggct ggcactgctg ctgctggcta gctctactgg gaaataaatt aggtgccttt
     2401 gaatcccagc aggcctcaga taaggaggga gaagctgcgc tgtggggctc ttgtcataga
     2461 ctgctggttt cttgcctccc ctgcccatag ctctattttt attccccacc ttgtgagaag
     2521 ctattctaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     2581 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     2641 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     2701 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2761 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2821 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2881 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2941 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3001 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3061 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3121 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3181 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3241 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3301 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3361 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3421 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3481 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3541 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3601 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3661 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3721 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3781 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3841 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3901 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3961 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4021 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4081 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4141 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4201 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4261 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4321 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4381 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4441 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4501 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4561 tactcatact cttccttttt caattcagaa gaactcgtca agaaggcgat agaaggcgat
     4621 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     4681 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     4741 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     4801 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     4861 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     4921 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     4981 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     5041 tactttctcg gcaggagcaa ggtgggatga caggagatcc tgccccggca cttcgcccaa
     5101 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     5161 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     5221 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     5281 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     5341 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     5401 ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
     5461 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc
     5521 gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac
     5581 ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat
     5641 ccggggtcag caccgtttct gcggactggc tttctacgtg ttccgcttcc tttagcagcc
     5701 cttgcgccct gaattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5761 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5821 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5881 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5941 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     6001 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     6061 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     6121 gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
     6181 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     6241 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     6301 cggccagtga attgtaatac gactcactat agggcgaatt gggccctcta gaagaatagc
     6361 ttctcacaag gtgggcaggg caggaatcct ggacatgcat atgtgtacac agttagtgct
     6421 aggtaagggt aagtctcatt taacagacaa ggagactgaa gatcaggcag ctgagtggct
     6481 tgctctagtc actccttcat taagggccca agccagctct tcacctaact atctatttca
     6541 gagcctggcc tgtcaccatc aggagaagca accagctctg gccgttccac tcctctacag
     6601 agcctggctc tagccatctt ggctctccgt catggaacta cttgttgggg gtttcctcta
     6661 ggtgggcgaa aatatctatt tactctgcca cgaagcgagt tcccctggtg agtctctaac
     6721 tgctaaacga cttgtcctgt cttgttgcag ggaagccgcc cactgggaaa catgagccac
     6781 tctgcaaagg aatatttaag cagtgttgtc acggcataca ctaacacaag t
//
