LOCUS       sequence-9824-ex        6180 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 6180)
  AUTHORS   Teruel MN, Blanpied TA, Shen K, Augustine GJ, Meyer T
  TITLE     A versatile microporation technique for the transfection of cultured
            CNS neurons.
  JOURNAL   J Neurosci Methods. 1999 Oct 30. 93(1):37-48.
  PUBMED    10598863
REFERENCE   2  (bases 1 to 6180)
  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 Neurosci 
            Methods. 1999 Oct 30. 93(1):37-48."
COMMENT     Sequence Label: PM-GFP
FEATURES             Location/Qualifiers
     source          1..6180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           896..938
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(994..996,997..999,1000..1710)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(994..996,997..999,1000..1710)
                     /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(1039..1060)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1300..1319)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1647..1668
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1732..1750)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1733..1750)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1770..1787)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1776..2000
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2046..2474
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2133..2152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2343..2364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2483..2503)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2488..2818
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2669..2804
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2731..2750
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2885..3679
                     /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(2939..2958)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3549..3568
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3853..3974
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3890..3909)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3944..3963
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4023..4039)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4023..4039)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4036..4058)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4047..4063
                     /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(4071..4077,4078..4095,4096..4101))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4116..4137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4254..4271)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4425..5013)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4505..4524)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5184..5975,5976..6044))
                     /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     5807..5826
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6045..6149)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcttgt
      901 tctttttgca gaagctcaga ataaacgctc aactttggca gatctaccat gggatgtatt
      961 aaatcaaaaa ggaaagacgg ggcccggtac catatggtga gcaagggcga ggagctgttc
     1021 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1081 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1141 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1201 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1261 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1321 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1381 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1441 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1501 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1561 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1621 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1681 atcactctcg gcatggacga gctgtacaag ggatcctcta gagggcccta ttctatagtg
     1741 tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1801 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1861 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1921 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     1981 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2041 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2101 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2161 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg ggcatccctt tagggttccg
     2221 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2281 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2341 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2401 tttataaggg attttgggga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2461 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2521 tccccaggca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2581 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2641 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2701 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     2761 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     2821 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     2881 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     2941 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3001 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3061 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3121 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3181 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3241 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3301 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3361 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     3421 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3481 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3541 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3601 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3661 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     3721 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3781 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3841 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3901 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3961 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     4021 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4081 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4141 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4201 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4261 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4321 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4381 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4441 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4501 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4561 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     4621 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4681 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4741 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4801 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4861 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4921 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4981 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5041 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5101 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5161 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5221 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5281 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5341 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5401 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5461 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5521 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5581 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5641 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5701 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5761 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5821 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5881 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5941 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6001 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6061 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6121 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
