LOCUS       sequence-189670-        6227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HR donor for fusing a p2A-mCherry reporter to mouse Actb. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6227)
  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 6227)
  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_Actb HR donor
FEATURES             Location/Qualifiers
     source          1..6227
                     /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             1055..1111
                     /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             1112..1822
                     /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(1257..1275)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1525..1544
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2660..2679)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2661..2679)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2686..2703)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2686..2702)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2695..2717)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2843..3271
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2930..2949)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3140..3161
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3615..4409
                     /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(3669..3688)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4279..4298
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4427..4495,4496..5287)
                     /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(4645..4664)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5458..6046
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5947..5966
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6200..6217
                     /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 gtaccccggg acctgacaga ctacctcatg aagatcctga ccgagcgtgg ctacagcttc
      301 accaccacag ctgagaggga aatcgtgcgt gacatcaaag agaagctgtg ctatgttgct
      361 ctagacttcg agcaggagat ggccactgcc gcatcctctt cctccctgga gaagagctat
      421 gagctgcctg acggccaggt catcactatt ggcaacgagc ggttccgatg ccctgaggct
      481 cttttccagc cttccttctt gggtaagttg tagcctagtc ctttctccat ctaaaggtga
      541 caaaactcct gaggccatag tacaagttaa gtctgatttc tgtcactctt ctcttaggta
      601 tggaatcctg tggcatccat gaaactacat tcaattccat catgaagtgt gacgttgaca
      661 tccgtaaaga cctctatgcc aacacagtgc tgtctggtgg taccaccatg tacccaggca
      721 ttgctgacag gatgcagaag gagattactg ctctggctcc tagcaccatg aagatcaagg
      781 taagctaagc atccttagct tggtgagggt gggccctgtg gttgtcagag caaccttcta
      841 ggtttaaggg gaatcccagc acccagagag ctcaccattc accatcttgt cttgctttct
      901 tcagatcatt gctcctcctg agcgcaagta ctctgtgtgg atcggtggct ccatcctggc
      961 ctcactgtcc accttccagc agatgtggat cagcaagcag gagtacgatg agtccggccc
     1021 ctccatcgtg caccgcaagt gcttcttaat taacgccact aacttctccc tgttgaaaca
     1081 agcaggggat gtcgaagaga atcccgggcc aatggtgagc aagggcgagg aggataacat
     1141 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1201 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     1261 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     1321 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     1381 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     1441 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     1501 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     1561 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     1621 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     1681 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     1741 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     1801 catggacgag ctgtacaagt aaggcgcgcc gcggactgtt actgagctgc gttttacacc
     1861 ctttctttga caaaacctaa cttgcgcaga aaaaaaaaaa ataagagaca acattggcat
     1921 ggctttgttt ttttaaattt tttttaaagt tttttttttt tttttttttt tttttttaag
     1981 tttttttgtt ttgttttggc gcttttgact caggatttaa aaactggaac ggtgaaggcg
     2041 acagcagttg gttggagcaa acatccccca aagttctaca aatgtggctg aggactttgt
     2101 acattgtttt gttttttttt ttttttggtt ttgtcttttt ttaatagtca ttccaagtat
     2161 ccatgaaata agtggttaca ggaagtccct caccctccca aaagccaccc ccactcctaa
     2221 gaggaggatg gtcgcgtcca tgccctgagt ccaccccggg gaaggtgaca gcattgcttc
     2281 tgtgtaaatt atgtactgca aaaatttttt taaatcttcc gccttaatac ttcatttttg
     2341 tttttaattt ctgaatggcc caggtctgag gcctcccttt tttttgtccc cccaacttga
     2401 tgtatgaagg ctttggtctc cctgggaggg ggttgaggtg ttgaggcagc cagggctggc
     2461 ctgtacactg acttgagacc aataaaagtg cacaccttac cttacacaaa cagcttgtgg
     2521 ctctgtggct ttgctgggtg tggggagcag gttgggtggg tgtggagctc tattgggggg
     2581 ggcatctagg gtgggctagg ccttgctgat ggtatctagt gggagggctc tcgagcatgc
     2641 atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
     2701 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2761 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2821 gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2881 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2941 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3001 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3061 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3121 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3181 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3241 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa ttcagggcgc aagggctgct
     3301 aaaggaagcg gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg
     3361 tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt
     3421 gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg
     3481 aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg
     3541 ctttcttgcc gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat
     3601 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3661 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3721 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3781 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3841 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3901 aagtgccggg gcaggatctc ctgtcatccc accttgctcc tgccgagaaa gtatccatca
     3961 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4021 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4081 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4141 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4201 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4261 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4321 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4381 tctatcgcct tcttgacgag ttcttctgaa ttgaaaaagg aagagtatga gtattcaaca
     4441 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4501 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4561 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4621 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4681 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4741 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4801 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4861 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4921 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4981 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5041 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5101 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5161 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5221 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5281 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5341 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5401 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5461 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5521 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5581 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5641 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5701 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5761 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5821 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5881 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5941 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6001 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6061 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6121 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6181 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaag
//
