LOCUS       sequence-313573-        7642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7642)
  AUTHORS   Bennardo N, Cheng A, Huang N, Stark JM
  TITLE     Alternative-NHEJ is a mechanistically distinct pathway of mammalian 
            chromosome break repair.
  JOURNAL   PLoS Genet. 2008 Jun 27;4(6):e1000110. doi: 
            10.1371/journal.pgen.1000110.
  PUBMED    18584027
REFERENCE   2  (bases 1 to 7642)
  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: "PLoS 
            Genet."; date: "2008-06-27"; volume: "4(6):e1000110. doi"; pages: " 
            10.1371/journal.pgen.1000110"
COMMENT     Sequence Label: EJ2GFP-puro
FEATURES             Location/Qualifiers
     source          1..7642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          658..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1763..1783
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1805..1825
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2074..2083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2080..2082,2083..2085,2086..2799)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2080..2082,2083..2085,2086..2796)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2125..2146)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2386..2405)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2733..2754
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3004..3023)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3069..3124
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3123..3142)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(3482..3500)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3483..3500)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3581..3600)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3700..3722
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3760..3778)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3846..3950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3951..4019,4020..4811)
                     /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(4169..4188)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4982..5570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5471..5490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5724..5741
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5828..5847
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5828..5846
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5884..6381
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5919..5942)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6314..6333
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6400..6999
                     /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(6400..6419)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6896..6916
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gsgcgcgccr gggggggggg gggggggggg gggggggggs
      541 ggggsgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      601 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      661 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      721 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      781 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      841 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg
      901 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
      961 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1081 gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggrggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattaat tcagatccgc
     1741 cgccactatg ggatcaagat cgccaaaaaa gaagagaaag gtgccgaaga agcatgcagc
     1801 accaccaaaa aaaaaacgaa aagtagaaga cccacgaggc aacaccagcg gcgtgctgag
     1861 cacccccaag gccaagaggg ccaagcaccc ccccggcacc gagaagccca ggagcaggag
     1921 ccagagcgag cagcccgcca cctgccccat ctgctacgcc gtgatcaggc agagcaggaa
     1981 cctgaggagg cacctggagc tgaggcactt cgccaagccc gcctagggat aacagggtaa
     2041 ttagatgaca agcccggcgt ggatccaccg gtcgccacca tggtgagcaa gggcgaggag
     2101 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2161 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     2221 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2281 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2341 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2401 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2461 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2521 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2581 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2641 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2701 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2761 gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagatca
     2821 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     2881 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taattcactc ctcaggtgca
     2941 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     3001 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     3061 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     3121 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     3181 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
     3241 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     3301 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     3361 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     3421 catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc tggcgtcgag
     3481 ttctatagtg tcacctaaat cgtatgtgta tgatacataa ggttatgtat taattgtagc
     3541 cgcgttctaa cgacaatatg tccatatggt gcactctcag tacaatctgc tctgatgccg
     3601 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3661 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3721 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     3781 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3841 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3901 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3961 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4021 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4081 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4141 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4201 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4261 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4321 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4381 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4441 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4501 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4561 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4621 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4681 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4741 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4801 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4861 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4921 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4981 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5041 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5101 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5161 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5221 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5281 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5341 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5401 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5461 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5521 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5581 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5641 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5701 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     5761 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcaggtta acctggctta
     5821 tcgaaattaa tacgactcac tatagggaga ccggcagatc tgatatcatc gatgaattct
     5881 accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc
     5941 tgggcacttg gcgctacaca agtggcctct ggcctcacac attccacatc caccggtagg
     6001 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
     6061 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
     6121 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
     6181 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
     6241 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
     6301 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
     6361 tcctcatctc cgggcctttc gaccgatcca gccgccacca tgaccgagta caagcccacg
     6421 gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc
     6481 gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc
     6541 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     6601 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     6661 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     6721 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     6781 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     6841 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     6901 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     6961 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag
     7021 cgcccgaccg aaaggagcgc acgaccccat ggctccgacc gaagccaccc ggggcggccc
     7081 cgccgacccc gcacccgccc ccgaggccca ccgcggggga cacaccgaac acgccgaccc
     7141 tgctgaacac gcggcgcagt tcggtgccca ggagcggatc gaaattgatg atctattaaa
     7201 caataaagat gtccactaaa atggaagttt ttcctgtcat actttgttaa gaagggtgag
     7261 aacagagtac ctacattttg aatggaagga ttggagctac gggggtgggg gtggggtggg
     7321 attagataaa tgcctgctct ttactgaagg ctctttacta ttgctttatg ataatgtttc
     7381 atagttggat atcataattt aaacaagcaa aaccaaatta agggccagct cattcctccc
     7441 actcatgatc tatagatcta tagatctctc gtgggatcat tgtttttctc ttgattccca
     7501 ctttgtggtt ctaagtactg tggtttccaa atgtgtcagt ttcatagcct gaagaacgag
     7561 atcagcagcc tctgttccac atacacttca ttctcagtat tgttttgcca agttctaatt
     7621 ccatcagaag cttcagctgc tc
//
