LOCUS       sequence-354452-        5395 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 5395)
  AUTHORS   Manderson AP, Kay JG, Hammond LA, Brown DL, Stow JL
  TITLE     Subcompartments of the macrophage recycling endosome direct the 
            differential secretion of IL-6 and TNFalpha.
  JOURNAL   J Cell Biol. 2007 Jul 2. 178(1):57-69.
  PUBMED    17606866
REFERENCE   2  (bases 1 to 5395)
  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: "J Cell 
            Biol. 2007 Jul 2. 178(1):57-69."
COMMENT     Sequence Label: GFP-TNFalpha
FEATURES             Location/Qualifiers
     source          1..5395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(3..360)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      17..152
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        169..360
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     317..337
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(362..466)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      493..948
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(580..599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     790..811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    955..1076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(966..985)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1020..1039
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1930..2640,2641..2643,2644..2646))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1930..2640,2641..2643,2644..2646))
                     /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(1972..1993)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2321..2340
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2580..2601
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2643..2652
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2691..2894)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2720..2740)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2895..3198
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3373..3961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3453..3472)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4290..4337
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4362..4381
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4569..5363)
                     /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             complement(4569..5363)
                     /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(4680..4699)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5290..5309
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tctttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag
       61 gccgaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa
      121 tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt
      181 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
      241 ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
      301 cctggggact ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag
      361 gactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
      421 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
      481 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      541 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      781 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
      841 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
      901 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttac gcgttaagat
      961 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1021 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     1081 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     1141 gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcagttatct agatccggtg
     1201 gatcccgggt ctcagagcaa tgactccaaa gtagacctgc ccggactccg caaagtctaa
     1261 gtacttgggc agattgacct cagcgctgag ttggtccccc ttctccagct ggaagactcc
     1321 tcccaggtat atgggttcat accagggttt gagctcagcc ccctcagggg tgtccttggg
     1381 gcaggggctc ttgacggcag agaggaggtt gactttctcc tggtatgaga tagcaaatcg
     1441 gctgacggtg tgggtgagga gcacgtagtc ggggcagcct tgtcccttga agagaacctg
     1501 ggagtagaca aggtacaacc catcggctgg caccactagt tggttgtctt tgagatccat
     1561 gccgttggcc aggagggcgt tggcgcgctg gctcagccac tccagctgct cctccacttg
     1621 gtggtttgct acgacgtggg ctacaggctt gtcactcgaa ttttgagaag atgatctgag
     1681 tgtgagggtc tgggccatag aactgatgag agggaggcca tttgggaact tctcatccct
     1741 ttggggaccg atcaccccga agttcagtag acagaagagc gtggtggccc ctgccacaag
     1801 caggaatgag aagaggctga gacataggca ccgcctggag ttctggaagc cccccatctt
     1861 ttgggggagt gcctcttctg ccagttccac gtcgcggatc atgctttctg tgctagatct
     1921 gagtccggac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc
     1981 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct
     2041 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
     2101 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt
     2161 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc
     2221 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
     2281 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc
     2341 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
     2401 gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt
     2461 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag
     2521 cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac
     2581 gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct
     2641 caccatggtg gcgaccggta gcgctagcgg atctgacggt tcactaaacc agctctgctt
     2701 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta
     2761 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
     2821 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
     2881 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
     2941 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     3001 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     3061 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     3121 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     3181 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     3241 gattactatt aataactaat gcatggcggt aatacggtta tccacagaat caggggataa
     3301 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3361 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3421 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3481 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3541 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3601 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3661 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3721 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3781 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3841 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3901 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3961 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4021 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4081 atgaagtttt aaatcaatct aaagtatata tgagtaacct gaggctatgg cagggcctgc
     4141 cgccccgacg ttggctgcga gccctgggcc ttcacccgaa cttggggggt ggggtgggga
     4201 aaaggaagaa acgcgggcgt attggcccca atggggtctc ggtggggtat cgacagagtg
     4261 ccagccctgg gaccgaaccc cgcgtttatg aacaaacgac ccaacaccgt gcgttttatt
     4321 ctgtcttttt attgccgtca tagcgcgggt tccttccggt attgtctcct tccgtgtttc
     4381 agttagcctc cccctagggt gggcgaagaa ctccagcatg agatccccgc gctggaggat
     4441 catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt
     4501 ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag
     4561 agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     4621 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     4681 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     4741 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     4801 tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc gaacagttcg
     4861 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     4921 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     4981 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     5041 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     5101 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     5161 gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc ggtcttgaca
     5221 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     5281 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     5341 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcga
//
