LOCUS       sequence-12494-e        6025 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 6025)
  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 6025)
  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: pIBB-mEGFP-IRES-puro2b
FEATURES             Location/Qualifiers
     source          1..6025
                     /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"
     RBS             638..649
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(845..847,848..850,851..1564)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MXSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(845..847,848..850,851..1561)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MXSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(890..911)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1151..1170)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1498..1519
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          1634..1863
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1846..1865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1921..2495,2496..2498,2499..2507)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2088..2105)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2315..2334
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2527..3126
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     complement(2527..2546)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3023..3043
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3262..3279)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3268..3492
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3539..3558
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3618..3634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3618..3634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3631..3653)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3642..3658
                     /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(3666..3672,3673..3690,3691..3696))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3711..3732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3849..3866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4020..4608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4100..4119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4779..5570,5571..5639))
                     /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     5402..5421
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5640..5744)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5819..5838)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6010..6025,1..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 atgggtggac 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 attcattaaa gaggagaaat taagcatgcg
      661 cacgaacgag aatgctaata caccagctgc ccgtcttcac agattcaaga acaagggaaa
      721 agacagtaca gaaatgaggc gtcncagaat agaggtcaat gtggagctga ggaaagctaa
      781 gaaggatgac cagatgctga agaggagaaa tgtaagctca tttcctgatg atgctacttc
      841 tccnatgnnn agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      901 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      961 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1021 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1081 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1141 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1201 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1261 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1321 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1381 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1441 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1501 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1561 gtaaagcggc cgcatagata actgatccag tgtgctggaa ttaattcgct gtctgcgagg
     1621 gccagctgtt ggggtgagta ctccctctca aaagcgggca tgacttctgc gctaagattg
     1681 tcagtttcca aaaacgagga ggatttgata ttcacctggc ccgcggtgat gcctttgagg
     1741 gtggccgcgt ccatctggtc agaaaagaca atctttttgt tgtcaagctt gaggtgtggc
     1801 aggcttgaga tctggccata cacttgagtg acaatgacat ccactttgcc tttctctcca
     1861 caggtgtcca ctcccaggtc caactgcagg tcgagcatgc atctagggcg gccaattccg
     1921 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     1981 tgcgtttgtc tatatgtgat tttccaccat attgccgtct tttggcaatg tgagggcccg
     2041 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     2101 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     2161 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     2221 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     2281 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     2341 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     2401 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     2461 ggacgtggtt ttcctttgaa aaacacgatg ataagcttgc cacaacccac aaggagacga
     2521 ccttccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg
     2581 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac
     2641 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     2701 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     2761 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     2821 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     2881 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     2941 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     3001 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     3061 accgccgacg tcgagtgccc gaaggaccgc gcgacctggt gcatgacccg caagcccggt
     3121 gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatggc
     3181 tccgaccgaa gccgacccgg gcggccccgc cgaccccgca cccgcccccg aggcccaccg
     3241 actctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     3301 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     3361 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     3421 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3481 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgagtgca ttctagttgt
     3541 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     3601 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3661 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3721 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3781 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3841 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3901 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3961 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4021 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4081 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4141 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4201 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4261 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4321 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4381 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4441 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4501 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4561 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4621 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4681 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4741 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4801 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4861 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4921 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4981 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5041 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5101 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5161 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5221 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5281 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5341 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5401 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5461 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5521 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5581 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5641 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5701 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5761 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtcg actctcagta
     5821 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     5881 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     5941 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     6001 atacgcgttg acattgatta ttgac
//
