LOCUS       sequence-178928-        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"
     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             1063..1119
                     /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             1120..1830
                     /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(1265..1283)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1533..1552
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1831..2419
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1884..1904)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2302..2313)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3264..3283)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3265..3283)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3290..3307)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3290..3306)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3299..3321)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3447..3875
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3534..3553)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3744..3765
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4219..5013
                     /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(4273..4292)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4883..4902
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(5031..5099,5100..5891)
                     /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(5249..5268)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6062..6650
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6551..6570
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6804..6821
                     /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 gccaagctta
      241 gaatagcttc tcacaaggtg gggaataaaa atagagctat gggcagggga ggcaagaaac
      301 cagcagtcta tgacaagagc cccacagcgc agcttctccc tccttatctg aggcctgctg
      361 ggattcaaag gcacctaatt tatttcccag tagagctagc cagcagcagc agtgccagcc
      421 cagagtgctg gtcagcagac cccgcctgct gtgttaggca ggggaggcag gcactgcagc
      481 agaaggtgaa cagacctttc ctggcagcgt ttgggtaccg gattaattgc taacccaaac
      541 acccatgcca tggggagact tgttcatccc agagcagttt gaggcaccgc cggatatggt
      601 tagatcttgg cttttgcaga cgccttgttg gacagtggct aaacccacgg cctgcctcca
      661 aagactctga tcctttcctc ggagtataca gttgagtgca ggcatggagg tggagacgtc
      721 tggagggctt caggttgctg acagagaaca caacagccta gggaagggac gtggatcctc
      781 cggtgctaac tctcaaaggc tgacttggaa gaggtgtgga gtgttcagca tgggagaaat
      841 gattttaagg acaagaatcc aaccagttag ggagatagca gtttgcttgg cccctccagc
      901 ctcggcttac cttctgctcc ttccttacag ggtgagtgga atctgcagcc tttgcctaag
      961 atcacctcca cgctggaaga acccacccca cgctgcccca tccgacagac tcagagcccc
     1021 gctaacccca ctgtgcccat cacaacagag gcagatgctg aagccactaa cttctccctg
     1081 ttgaaacaag caggggatgt cgaagagaat cccgggccaa tggtgagcaa gggcgaggag
     1141 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     1201 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     1261 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     1321 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     1381 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1441 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     1501 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     1561 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     1621 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     1681 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     1741 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     1801 accggcggca tggacgagct gtacaagtaa aatcaacctc tggattacaa aatttgtgaa
     1861 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     1921 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     1981 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     2041 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     2101 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     2161 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     2221 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     2281 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     2341 ctgccggctc tgcggcctct tccgcgtctc cgccttcgcc ctcagacgag tcggatctcc
     2401 ctttgggccg cctccccgcc acttgtgaga agctattctt cagaccctcc tcctcctgtc
     2461 cacctgggct cacaccagct ctgtgtagcc ctcccttcct gtgtgaagac tcacttccat
     2521 agactagtgc tggccatcca gggagaagga gcatgtcacc ttggagacac ttgcatgact
     2581 gcaccccatg aggtccaact gaacggagaa gccctggaca ttatctacac actgctgact
     2641 ccacataggg acagctcgct gtggtagtcc agattcaaca ccaccttgcc tccagaacag
     2701 ccttgccagg ctgggtgcag actccccaag ccccacagtc ccgaccctgt tggtcctctg
     2761 gtggtgactt gtgttagtgt atgccgtgac aacactgctt aaatattcct ttgcagagtg
     2821 gctcatgttt cccagtgggc ggcttccctg caacaagaca ggacaagtcg tttagcagtt
     2881 agagactcac caggggaact cgcttcgtgg cagagtaaat agatattttc gcccacctag
     2941 aggaaacccc caacaagtag ttccatgacg gagagccaag atggctagag ccaggctctg
     3001 tagaggagtg gaacggccag agctggttgc ttctcctgat ggtgacaggc caggctctga
     3061 aatagatagt taggtgaaga gctggcttgg gcccttaatg aaggagtgac tagagcaagc
     3121 cactcagctg cctgatcttc agtctccttg tctgttaaat gagacttacc cttacctagc
     3181 actaactgtg tacacatatg catgtccagg attcctgccc tgcccacctt gtgagaagct
     3241 attcttctag agggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg
     3301 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     3361 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     3421 agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg
     3481 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3541 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3601 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3661 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3721 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3781 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3841 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaattcagg gcgcaagggc
     3901 tgctaaagga agcggaacac gtagaaagcc agtccgcaga aacggtgctg accccggatg
     3961 aatgtcagct actgggctat ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta
     4021 gcttgcagtg ggcttacatg gcgatagcta gactgggcgg ttttatggac agcaagcgaa
     4081 ccggaattgc cagctggggc gccctctggt aaggttggga agccctgcaa agtaaactgg
     4141 atggctttct tgccgccaag gatctgatgg cgcaggggat caagatctga tcaagagaca
     4201 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4261 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4321 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4381 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     4441 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4501 ggcgaagtgc cggggcagga tctcctgtca tcccaccttg ctcctgccga gaaagtatcc
     4561 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4621 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4681 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4741 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4801 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4861 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4921 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4981 gccttctatc gccttcttga cgagttcttc tgaattgaaa aaggaagagt atgagtattc
     5041 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5101 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5161 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5221 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5281 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5341 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5401 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5461 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5521 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5581 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5641 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5701 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5761 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5821 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5881 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5941 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6001 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6061 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6121 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6181 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6241 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6301 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6361 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6421 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6481 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6541 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6601 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6661 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6721 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6781 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
