LOCUS       sequence-66699-e        6220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP in Promega pCIneo (uses CMV promoter). Superior to pfwB for some
            purposes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  AUTHORS   Buck CB, Thompson CD
  TITLE     Production of papillomavirus-based gene transfer vectors.
  JOURNAL   Curr Protoc Cell Biol. 2007 Dec;Chapter 26:Unit 26.1. doi: 
            10.1002/0471143030.cb2601s37.
  PUBMED    18228512
REFERENCE   2  (bases 1 to 6220)
  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: "Curr Protoc
            Cell Biol."; date: "2007-12"; volume: "Chapter 26:Unit 26.1. doi"; 
            pages: " 10.1002/0471143030.cb2601s37"
COMMENT     Sequence Label: pCIneoEGFP
FEATURES             Location/Qualifiers
     source          1..6220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1091..1143
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1151..1160
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1157..1159,1160..1162,1163..1876)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1157..1159,1160..1162,1163..1873)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1202..1223)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1463..1482)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1810..1831
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1924..2045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1935..1954)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1989..2008
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      2231..2686
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2318..2337)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2528..2549
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2803..3160
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2803..2994
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2826..2846)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3011..3146
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3211..4005
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3265..3284)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3875..3894
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4069..4117
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4158..4177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4277..4299
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4337..4355)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4423..4527
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4528..4596,4597..5388)
                     /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     complement(4746..4765)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5559..6147
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6048..6067
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga
     1141 tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1201 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1261 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1321 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1381 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1441 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1501 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1561 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1621 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1681 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1741 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1801 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1861 cgagctgtac aagtaaagcg gccgcttccc tttagtgagg gttaatgctt cgagcagaca
     1921 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     1981 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     2041 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     2101 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat ccgataagga tcgatccggg
     2161 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     2221 tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2281 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2341 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2401 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2461 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2521 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2581 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2641 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt
     2701 ttctccttac gcatctgtgc ggtatttcac accgcatacg cggatctgcg cagcaccatg
     2761 gcctgaaata acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca
     2821 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2881 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2941 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3001 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3061 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3121 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagcttgatt cttctgacac
     3181 aacagtctcg aacttaaggc tagagccacc atgattgaac aagatggatt gcacgcaggt
     3241 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3301 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3361 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     3421 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3481 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     3541 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3601 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3661 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3721 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3781 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3841 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3901 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3961 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4021 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg atggccgcaa taaaatatct
     4081 ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagcgat aaggatccgc
     4141 gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4201 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4261 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4321 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4381 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4441 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4501 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4561 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4621 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4681 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4741 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4801 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4861 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4921 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4981 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5041 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5101 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5161 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5221 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5281 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5341 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5401 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5461 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5521 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5581 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5641 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5701 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5761 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5821 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5881 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5941 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6001 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6061 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6121 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6181 gccttttgct ggccttttgc tcacatggct cgacagatct
//
