LOCUS       sequence-65152-e        7990 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 7990)
  TITLE     Craig lab unpublished plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7990)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pEGFPC1/GgVcl 1-1066 A50I K1306A, R1308A mutant
FEATURES             Location/Qualifiers
     source          1..7990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4778..4899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4815..4834)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4869..4888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4906..5361)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5043..5064)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5255..5274
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5388..5492
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5494..5851
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5494..5685
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5517..5537)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5702..5837
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5886..6680
                     /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             5886..6680
                     /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(5940..5959)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6550..6569
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6868..6887)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6912..6959
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7288..7876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7777..7796
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attccggccc cccctgcccc
     1381 gccgccgcca tgcccgtctt ccacacgcgc accatcgaga gcatcttgga gcccgtggct
     1441 cagcagatct cccacctggt catcatgcac gaggaggggg aggtagacgg caaggccatc
     1501 ccggacctca ccgcccccgt gtcggccgtg caggccatcg tcagcaacct ggtgcgggtt
     1561 ggaaaagaaa ctgtgcagac aacagaagac cagatcttga aaagggatat gccaccagca
     1621 ttcatcaaag tagagaatgc ctgcaccaag ctcgttcgag cagcccagat gctgcaagca
     1681 gatccttatt cagtaccagc tcgtgactac ctaattgatg gatcaagagg catcctttct
     1741 ggaacatcag acttacttct gacatttgat gaagcggagg tccgtaaaat catccgtgtc
     1801 tgcaaaggaa tattggaata tctgactgtg gcagaagtag tagagactat ggaggatttg
     1861 gtgacatata caaagaatct agggccagga atgacaaaga tggcgaaaat gatcgatgag
     1921 agacaacagg aattaactca tcaggaacat agggttatgc tggtgaactc catgaatact
     1981 gtgaaggagc tattgcctgt acttatttca gctatgaaga tctttgtaac cacaaaaaac
     2041 actaaaagcc agggaataga agaggccttg aaaaatcgca atttcacagt agagaaaatg
     2101 agtgctgaga taaatgaaat aatccgtgta ttacaactca cttcctggga tgaagatgcc
     2161 tgggccagca aggacactga agccatgaaa agagctttag ccctaataga ttcaaagatg
     2221 aaccaggcaa aaggctggct gagagatcca aacgcacctc caggggatgc tggtgagcaa
     2281 gcaataaggc agatccttga tgaagctgga aaagcaggag aattgtgtgc aggcaaagaa
     2341 cgcagggaga ttctgggaac atgcaaaact ttgggccaaa tgactgatca acttgctgat
     2401 ctccgagcta gaggacaggg tgctacaccg atggcaatgc agaaggcaca gcaggtgtca
     2461 caaggcctgg atttgctcac tgcaaaagtg gagaatgcag cccggaaatt ggaggccatg
     2521 acaaactcta agcaggctat tgcaaagaag attgatgctg ctcagaactg gcttgcggat
     2581 cccaacggcg gaagtgaagg agaagaacac attcgaggaa ttatgtctga agcaaggaaa
     2641 gttgcagaat tgtgtgagga gcctaaagaa agagatgata tccttcgctc cttgggggaa
     2701 atctctgctc tgacagctaa gctgtcagat ctgcgacgac atgggaaagg cgactctcct
     2761 gaggcccgtg cattggccaa gcaaatagct acatcacttc agaacttaca gtccaaaaca
     2821 aacagagctg tagcaaatac taggccagtt aaagctgctg tccatttgga gggcaagatt
     2881 gagcaagccc agaggtggat agataatcct acagttgatg atcggggagt aggccaggca
     2941 gcaattcggg gtttggttgc agaaggtcgt cgtctagcca atgtcatgat gggaccttat
     3001 cgtcaggacc tgcttgccaa atgtgaccgt gtagaccagc tggctgctca gcttgctgac
     3061 cttgcagcaa gaggggaagg agagtctcct caggctaggg caattgctgc tcagcttcag
     3121 gactccctga aggatctcaa agcacggatg caagaagcaa tgacccagga ggtgtctgat
     3181 gttttcagtg acactacaac ccctattaag ttgttagcag tagcagccac tgctccttct
     3241 gatactccca atagagaaga ggtgtttgaa gaaagagcag caaattttga aaaccatgct
     3301 gctagactgg gagcaacagc agaaaaagca gctgcagttg gaactgctaa taaaactact
     3361 gtggaaggca ttcaggcaac agtcaaatct gcaagggagc ttacaccaca ggtagtatcg
     3421 gctgctcgaa tcctcctgag aaatcctgga aatcaagctg cttatgagca ttttgagaca
     3481 atgaaaaacc aatggattga taatgtagaa aagatgacag ggctggtgga tgaggccatc
     3541 gataccaagt ctctgttgga tgcatcagaa gaggctatta agaaggatct tgataaatgc
     3601 aaagttgcaa tggccaatat gcaacctcag atgctggtag ctggagccac cagcattgct
     3661 agacgagcga accgcatcct gcttgtggca aaacgggagg ttgaaaattc agaagaccct
     3721 aaattcaggg aggctgttaa agcagcttct gatgagctga gcaaaaccat atcaccgatg
     3781 gtaatggatg ctaaagctgt agcaggaaat atctctgatc ctggtttgca gaagagtttc
     3841 ttggattctg gatacaggat tctgggagct gtggccaaag tcagagaagc ctttcagcct
     3901 caggagccag actttccccc tcctcctcct gacctagagc atctccatct gactgatgag
     3961 cttgctcctc caaaaccacc acttccagaa ggtgaggttc ccccacccag accaccaccc
     4021 cctgaagaaa aagatgagga gttcccagag cagaaagcag gagaagctat taatcagccc
     4081 atgatgatgg ctgctaggca gttgcatgac gaggctcgga aatggtctag caagggtaac
     4141 gacatcattg ctgctgctaa acgaatggcg ctgctcatgg ctgagatgtc acgcctggtg
     4201 cgaggaggca gcggaaacaa gcgtgccctc atccagtgtg caaaagatat tgctaaggca
     4261 tcggatgaag tcactcgatt ggccaaagag gtggccaagc aatgtactga taaacgcatt
     4321 agaacaaacc tcttacaggt ctgtgagcga atcccaacca tcagcacgca gctcaaaatt
     4381 ctttccacag tgaaagctac catgctgggc aggactaaca tcagcgatga agaatcagaa
     4441 caggcaactg agatgttggt tcataacgcc cagaacctca tgcagtctgt gaaggaaact
     4501 gtgagagaag ctgaagcagc atccattaag ataagaacag atgccggatt cactctgcgc
     4561 tgggtcagaa agaccccatg gtatcagtaa acaccacata agcattgtct tctgtcggaa
     4621 ttctgcagtc gacggtaccg cgggcccggg atccaccgga tctagataac tgatcataat
     4681 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     4741 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     4801 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4861 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta
     4921 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4981 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     5041 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     5101 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     5161 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     5221 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     5281 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     5341 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5401 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5461 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     5521 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     5581 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     5641 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5701 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5761 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5821 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     5881 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     5941 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     6001 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     6061 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     6121 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     6181 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     6241 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     6301 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     6361 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     6421 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     6481 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     6541 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     6601 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     6661 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     6721 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     6781 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     6841 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     6901 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     6961 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     7021 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     7081 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     7141 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7201 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7261 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7321 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7381 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     7441 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7501 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7561 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7621 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7681 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7741 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7801 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7861 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7921 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7981 cgccatgcat
//
