LOCUS       sequence-257581-        5366 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5366)
  AUTHORS   Watanabe N, Mitchison TJ
  TITLE     Single-molecule speckle analysis of actin filament turnover in 
            lamellipodia.
  JOURNAL   Science. 2002 Feb 8. 295(5557):1083-6.
  PUBMED    11834838
REFERENCE   2  (bases 1 to 5366)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2002 Feb 8. 295(5557):1083-6."
COMMENT     Sequence Label: Reduced Expression GFP beta actin
FEATURES             Location/Qualifiers
     source          1..5366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      227..236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(233..235,236..238,239..949)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(233..235,236..238,239..949)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(278..299)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(539..558)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     886..907
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2228..2349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2265..2284)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2319..2338
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2356..2811)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2493..2514)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2705..2724
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2838..2942
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2944..3301
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2944..3135
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2967..2987)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3152..3287
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3336..4130
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3336..4130
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3390..3409)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4000..4019
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4318..4337)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4362..4409
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4738..5326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5227..5246
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
       61 ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      121 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctgggag gtctatataa
      181 gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggtcgcca ccatggtgag
      241 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
      301 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
      361 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
      421 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
      481 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
      541 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
      601 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
      661 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
      721 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
      781 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
      841 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
      901 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggactcag
      961 atctcgagct atggatgatg atatcgccgc gctcgtcgtc gacaacggct ccggcatgtg
     1021 caaggccggc ttcgcgggcg acgatgcccc ccgggccgtc ttcccctcca tcgtggggcg
     1081 ccccaggcac cagggcgtga tggtgggcat gggtcagaag gattcctatg tgggcgacga
     1141 ggcccagagc aagagaggca tcctcaccct gaagtacccc atcgagcacg gcatcgtcac
     1201 caactgggac gacatggaga aaatctggca ccacaccttc tacaatgagc tgcgtgtggc
     1261 tcccgaggag caccccgtgc tgctgaccga ggcccccctg aaccccaagg ccaaccgcga
     1321 gaagatgacc cagatcatgt ttgagacctt caacacccca gccatgtacg ttgctatcca
     1381 ggctgtgcta tccctgtacg cctctggccg taccactggc atcgtgatgg actccggtga
     1441 cggggtcacc cacactgtgc ccatctacga ggggtatgcc ctcccccatg ccatcctgcg
     1501 tctggacctg gctggccggg acctgactga ctacctcatg aagatcctca ccgagcgcgg
     1561 ctacagcttc accaccacgg ccgagcggga aatcgtgcgt gacattaagg agaagctgtg
     1621 ctacgtcgcc ctggacttcg agcaagagat ggccacggct gcttccagct cctccctgga
     1681 gaagagctac gagctgcctg acggccaggt catcaccatt ggcaatgagc ggttccgctg
     1741 ccctgaggca ctcttccagc cttccttcct gggcatggag tcctgtggca tccacgaaac
     1801 taccttcaac tccatcatga agtgtgacgt ggacatccgc aaagacctgt acgccaacac
     1861 agtgctgtct ggcggcacca ccatgtaccc tggcattgcc gacaggatgc agaaggagat
     1921 cactgccctg gcacccagca caatgaagat caagatcatt gctcctcctg agcgcaagta
     1981 ctccgtgtgg atcggcggct ccatcctggc ctcgctgtcc accttccagc agatgtggat
     2041 cagcaagcag gagtatgacg agtccggccc ctccatcgtc caccgcaaat gcttctaggg
     2101 atccaccgga tctagataac tgatcataat cagccatacc acatttgtag aggttttact
     2161 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2221 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2281 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2341 tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2401 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2461 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2521 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2581 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2641 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2701 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2761 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     2821 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2881 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2941 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3001 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3061 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3121 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3181 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3241 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3301 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3361 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3421 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3481 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3541 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3601 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3661 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3721 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3781 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3841 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     3901 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     3961 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4021 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4081 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4141 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4201 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4261 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     4321 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     4381 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     4441 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     4501 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     4561 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     4621 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4681 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4741 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4801 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4861 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4921 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4981 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5041 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5101 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5161 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5221 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5281 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5341 ggccttttta cggttcctgg cctttt
//
