LOCUS       sequence-417244-        8314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The HDR-based surrogate reporter for validating HDR-editing activity
            of different gene editing tools as well as for helping to screen 
            HDR-editing positive cells. Key Construct: CAG-PuroR-CMV-eGFP*(HDR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8314)
  AUTHORS   Shao S, Ren C, Liu Z, Bai Y, Chen Z, Wei Z, Wang X, Zhang Z, Xu K
  TITLE     Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian
            cells by expressing Saccharomyces cerevisiae Rad52.
  JOURNAL   Int J Biochem Cell Biol. 2017 Nov;92:43-52. doi: 
            10.1016/j.biocel.2017.09.012. Epub 2017 Sep 18.
  PUBMED    28928041
REFERENCE   2  (bases 1 to 8314)
  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: "Int J 
            Biochem Cell Biol."; date: "2017-11"; volume: "92:43-52. doi"; 
            pages: " 10.1016/j.biocel.2017.09.012. Epub 2017 Sep 18"
COMMENT     Sequence Label: pHDR-eGFP (pXK-1647)
FEATURES             Location/Qualifiers
     source          1..8314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(131..199,200..991)
                     /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(349..368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1162..1750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1651..1670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1904..1921
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2038..2059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2074..2079,2080..2097,2098..2104)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2112..2128
                     /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     2117..2139
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2136..2152
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2136..2152
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2173..2191
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   2340..2374
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        2753..3132
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3134..3411
                     /label=chicken beta-actin promoter
     intron          3412..4428
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4352..4374
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4436..4455
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4490..5089
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4490..4509)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4986..5006
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5093..5112)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5158..5213
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5212..5231)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     enhancer        5622..5925
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5926..6129
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6080..6100
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      6168..6177
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6219..6240)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6527..6546)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6874..6895
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    7076..7197
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7113..7132)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7167..7186
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7547..7609
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7675..7694)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7676..7694)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7701..7718)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7701..7717)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7710..7732)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7859..8287
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7946..7965)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8156..8177
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
       61 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
      121 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      181 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      241 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      301 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
      361 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
      421 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
      481 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
      541 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
      601 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
      661 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
      721 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
      781 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
      841 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
      901 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
      961 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     1021 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1081 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     1321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1741 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1801 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     1861 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     1921 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     1981 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     2041 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     2101 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     2161 cgccaagctc gaaattaacc ctcactaaag ggaacaaaag ctggtacctc gcgcgacttg
     2221 gtttgccatt ctttagcgcg cgtcgcgtca cacagcttgg ccacaatgtg gtttttgtca
     2281 aacgaagatt ctatgacgtg tttaaagttt aggtcgagta aagcgcaaat cttttttaac
     2341 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
     2401 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
     2461 cgtgaggcgt gcttgtcaat gcggtaagtg tcactgattt tgaactataa cgaccgcgtg
     2521 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
     2581 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
     2641 tagatatcgt gactaatata taataaaatg ggtagttctt tagacgatga gcatatcctc
     2701 tctgctcttc tgcaaagcga tgacgagctt gttggctagc ggtggcggcc tcgacattga
     2761 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     2821 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2881 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2941 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     3001 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     3061 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     3121 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     3181 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggsg
     3241 gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg
     3301 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     3361 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     3421 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     3481 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     3541 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
     3601 gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
     3661 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     3721 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
     3781 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
     3841 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc
     3901 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
     3961 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     4021 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
     4081 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
     4141 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     4201 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     4261 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     4321 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     4381 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     4441 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tggccgcgta tgaccgagta
     4501 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     4561 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     4621 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     4681 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     4741 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     4801 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     4861 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     4921 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     4981 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     5041 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa gatctttttc
     5101 cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact tctggctaat
     5161 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     5221 ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg
     5281 caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt
     5341 atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt
     5401 agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct
     5461 tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc
     5521 ctcttctctt atggagatcc ctcgacctgc agcccaagct ttagttatta atagtaatca
     5581 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     5641 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     5701 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     5761 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     5821 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     5881 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     5941 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     6001 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     6061 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     6121 agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtcgcc accatggtga
     6181 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     6241 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     6301 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     6361 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     6421 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcagcggc cgctcggcca
     6481 ccacagggaa gctggggatc ctaagtgact aaccatcttc ttcaaggacg acggcaacta
     6541 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     6601 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     6661 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     6721 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     6781 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     6841 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     6901 cgccgggatc actctcggca tggacgagct gtacaagtcc ggactcagat ccaccggatc
     6961 tagataactg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     7021 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     7081 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     7141 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttactc
     7201 gagttaatta acgagagcat aatattgata tgtgccaaag ttgtttctga ctgactaata
     7261 agtataattt gtttctatta tgtataggtt aagctaatta cttattttat aatacaacat
     7321 gactgttttt aaagtacaaa ataagtttat ttttgtaaaa gagagaatgt ttaaaagttt
     7381 tgttacttta tagaagaaat tttgagtttt tgtttttttt taataaataa ataaacataa
     7441 ataaattgtt tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat
     7501 ctattcaaat taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt
     7561 acgcatgatt atctttaacg tacgtcacaa tatgattatc tttctagggt taaataatag
     7621 tttctaattt ttttattatt cagcctgctg tcgtgaatac cgagctccaa ttcgccctat
     7681 agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     7741 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     7801 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     7861 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     7921 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     7981 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     8041 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     8101 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     8161 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     8221 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     8281 gcgaatttta acaaaatatt aacgcttaca attt
//
