LOCUS       sequence-220168-        6796 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 6796)
  AUTHORS   Held M, Schmitz MH, Fischer B, Walter T, Neumann B, Olma MH, Peter 
            M, Ellenberg J, Gerlich DW
  TITLE     CellCognition: time-resolved phenotype annotation in high-throughput
            live cell imaging.
  JOURNAL   Nat Methods. 2010 Sep . 7(9):747-54.
  PUBMED    20693996
REFERENCE   2  (bases 1 to 6796)
  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: "Nat 
            Methods. 2010 Sep . 7(9):747-54."
COMMENT     Sequence Label: pEGFP-PCNA-IRES-puro2b
FEATURES             Location/Qualifiers
     source          1..6796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    609..665
                     /label=MCS
                     /note="multiple cloning site"
     CDS             666..686
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      699..708
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(705..707,708..710,711..1421)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(750..771)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1011..1030)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1358..1379
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2280..2336
                     /label=MCS
                     /note="multiple cloning site"
     intron          2405..2634
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2617..2636
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2692..3266,3267..3269,3270..3278)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2859..2876)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3086..3105
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3298..3897
                     /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(3298..3317)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3794..3814
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4033..4050)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4039..4263
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4310..4329
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4389..4405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4389..4405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4402..4424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4413..4429
                     /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(4437..4443,4444..4461,4462..4467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4482..4503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4620..4637)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4791..5379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4871..4890)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5550..6341,6342..6410))
                     /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     6173..6192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6411..6515)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6590..6609)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6781..6796,1..364)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccccgaa gaagaagcgc aaagtactgg taccggtcgc caccatggtg agcaagggcg
      721 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
      781 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
      841 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
      901 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
      961 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1021 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1081 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1141 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggccaact
     1201 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1261 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1321 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1381 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcgaaggg caagggcaag
     1441 ggcaagggcc gggccgcggc tacgcgtatc gatccatgtt cgaggcgcgc ctggtccagg
     1501 gctccatcct caagaaggtg ttggaggcac tcaaggacct catcaacgag gcctgctggg
     1561 atattagctc cagcggtgta aacctgcaga gcatggactc gtcccacgtc tctttggtgc
     1621 agctcaccct gcggtctgag ggcttcgaca cctaccgctg cgaccgcaac ctggccatgg
     1681 gcgtgaacct caccagtatg tccaaaatac taaaatgcgc cggcaatgaa gatatcatta
     1741 cactaagggc cgaagataac gcggatacct tggcgctagt atttgaagca ccaaaccagg
     1801 agaaagtttc agactatgaa atgaagttga tggatttaga tgttgaacaa cttggaattc
     1861 cagaacagga gtacagctgt gtagtaaaga tgccttctgg tgaatttgca cgtatatgcc
     1921 gagatctcag ccatattgga gatgctgttg taatttcctg tgcaaaagac ggagtgaaat
     1981 tttctgcaag tggagaactt ggaaatggaa acattaaatt gtcacagaca agtaatgtcg
     2041 ataaagagga ggaagctgtt accatagaga tgaatgaacc agttcaacta acttttgcac
     2101 tgaggtacct gaacttcttt acaaaagcca ctccactctc ttcaacggtg acactcagta
     2161 tgtctgcaga tgtacccctt gttgtagagt ataaaattgc ggatatggga cacttaaaat
     2221 actacttggc tcccaagatc gaggatgaag aaggatctta gtctagcgct accggactca
     2281 gatctcgagc tcaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccgcgg
     2341 ccgcatagat aactgatcca gtgtgctgga attaattcgc tgtctgcgag ggccagctgt
     2401 tggggtgagt actccctctc aaaagcgggc atgacttctg cgctaagatt gtcagtttcc
     2461 aaaaacgagg aggatttgat attcacctgg cccgcggtga tgcctttgag ggtggccgcg
     2521 tccatctggt cagaaaagac aatctttttg ttgtcaagct tgaggtgtgg caggcttgag
     2581 atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc
     2641 actcccaggt ccaactgcag gtcgagcatg catctagggc ggccaattcc gcccctctcc
     2701 ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
     2761 ctatatgtga ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
     2821 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg
     2881 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc
     2941 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
     3001 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
     3061 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
     3121 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
     3181 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt
     3241 tttcctttga aaaacacgat gataagcttg ccacaaccca caaggagacg accttccatg
     3301 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc
     3361 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc
     3421 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     3481 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     3541 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     3601 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     3661 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     3721 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     3781 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     3841 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc
     3901 ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctccgaccga
     3961 agccacccgg ggcggccccg ccgaccccgc acccgccccc gaggcccacc gactctagag
     4021 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4081 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     4141 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4201 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     4261 tggcttctga ggcggaaaga accagctggg gctcgagtgc attctagttg tggtttgtcc
     4321 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     4381 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4441 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4501 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4561 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4621 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4681 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4741 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4801 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4861 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4921 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4981 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5041 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5101 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5161 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5221 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5281 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5341 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5401 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5461 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5521 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5581 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5641 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5701 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5761 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5821 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5881 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5941 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6001 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6061 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6121 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6181 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6241 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6301 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6361 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6421 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6481 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6541 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     6601 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     6661 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     6721 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     6781 gacattgatt attgac
//
