LOCUS       sequence-224007-        6133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6133)
  AUTHORS   Lai MB, Zhang C, Shi J, Johnson V, Khandan L, McVey J, Klymkowsky 
            MW, Chen Z, Junge HJ
  TITLE     TSPAN12 Is a Norrin Co-receptor that Amplifies Frizzled4 Ligand 
            Selectivity and Signaling.
  JOURNAL   Cell Rep. 2017 Jun 27;19(13):2809-2822. doi: 
            10.1016/j.celrep.2017.06.004.
  PUBMED    28658627
REFERENCE   2  (bases 1 to 6133)
  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"; doi: 
            "10.1016/j.celrep.2017.06"; journalName: "Cell Rep"; date: 
            "2017-06-27- 27"; volume: "19"; issue: "13"; pages: "2809-2822"
COMMENT     Sequence Label: pcDNA 3.3 EGFP
FEATURES             Location/Qualifiers
     source          1..6133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      742..751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(748..750,751..753,754..1467)
                     /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(748..750,751..753,754..1464)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(793..814)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1054..1073)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1401..1422
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1519..1538)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1563..1611
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1813..2241
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1900..1919)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2110..2131
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2250..2270)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2255..2584
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2435..2570
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2497..2516
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2651..3445
                     /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(2705..2724)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3315..3334
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3621..3742
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3658..3677)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3712..3731
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3791..3807)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3791..3807)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3804..3826)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3815..3831
                     /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(3839..3845,3846..3863,3864..3869))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3884..3905
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4022..4039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4193..4781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4273..4292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4952..5743,5744..5812))
                     /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     5575..5594
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5813..5917)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5992..6011)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tgccaccatg gtgagcaagg gcgaggagct gttcaccggg
      781 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
      841 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
      901 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
      961 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1021 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1081 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1141 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1201 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1261 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1321 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1381 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1441 ctcggcatgg acgagctgta caagtaaaag ggttcgatcc ctaccggtta gtaatgagtt
     1501 taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     1561 gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg
     1621 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     1681 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     1741 tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc tggggctcta
     1801 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1861 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1921 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1981 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2041 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2101 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2161 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2221 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2281 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2341 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2401 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     2461 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     2521 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     2581 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     2641 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2701 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2761 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     2821 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     2881 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     2941 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3001 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3061 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3121 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     3181 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3241 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3301 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3361 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3421 tcgccttctt gacgagttct tctgagcggg actctggggt tcgcgaaatg accgaccaag
     3481 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3541 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3601 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     3661 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3721 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     3781 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3841 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3901 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3961 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4021 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4081 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4141 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4201 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4261 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4321 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4381 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4441 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4501 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4561 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4621 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4681 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4741 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4801 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4861 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4921 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4981 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5041 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5101 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5161 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5221 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5281 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5341 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5401 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5461 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5521 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5581 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5641 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5701 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5761 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5821 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5881 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5941 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     6001 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     6061 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     6121 gaatctgctt agg
//
