LOCUS       sequence-8219-ex        7560 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 7560)
  AUTHORS   Slimko EM, Lester HA
  TITLE     Codon optimization of Caenorhabditis elegans GluCl ion channel genes
            for mammalian cells dramatically improves expression levels.
  JOURNAL   J Neurosci Methods. 2003 Mar 30. 124(1):75-81.
  PUBMED    12648766
REFERENCE   2  (bases 1 to 7560)
  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. 2003 Mar 30. 124(1):75-81."
COMMENT     Sequence Label: optGluCl beta Y182F ECFP
FEATURES             Location/Qualifiers
     source          1..7560
                     /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"
     regulatory      954..963
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2067..2069,2070..2072,2073..2783)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2112..2133)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2373..2392)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2720..2741
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3057..3098
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3108..3125
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3148..3165)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3154..3378
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3424..3852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3511..3530)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3721..3742
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3861..3881)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3866..4196
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4047..4182
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4109..4128
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4263..5057
                     /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(4317..4336)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4927..4946
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5233..5354
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5270..5289)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5324..5343
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5403..5419)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5403..5419)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5416..5438)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5427..5443
                     /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(5451..5457,5458..5475,5476..5481))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5496..5517
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5634..5651)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5805..6393)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5885..5904)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6564..7355,7356..7424))
                     /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     7187..7206
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7425..7529)
                     /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 gctggctagt
      901 taagcttggt accgagctcg gatccactag tccagtgtgg tggaattgcc cttgccacca
      961 tggccacccc ctccagcttc tcgatcctgc tgctgctgct gctgatgccc gtggtgacca
     1021 acggcgagta cagcatgcag agcgagcagg agatcctgaa tgccctgctg aagaactacg
     1081 acatgcgcgt gcgccccccc cccgccaaca gcagcaccga gggcgccgtg aacgtgagag
     1141 tgaacatcat gatcagaatg ctgagcaaga tcgacgtggt gaacatggag tactccatcc
     1201 agctgacctt ccgcgagcag tggatcgacc cccgcctggc ctacgagaac ctgggcttct
     1261 acaacccccc cgccttcctg accgtgcccc acgtcaagaa gtccctgtgg atacccgaca
     1321 ccttcttccc caccgagaag gccgcccacc gccacctgat cgacatggag aacatgttcc
     1381 tgcgcatcta ccccgacggc aagatcctgt acagcagccg catcagcctg acgagctcct
     1441 gccccatgcg cctgcagctg taccccctgg actaccagag ctgcaacttc gacctggtgt
     1501 ccttcgccca caccatgaac gacatcatgt atgagtggga ccccagcacc cccgtgcagc
     1561 tgaagcccgg cgtgggctcc gacctgccca acttcatcct gaagaactac accaccaacg
     1621 ccgactgcac ctcccacacc aacactggct cgtacggctg cctgcgcatg cagctgctgt
     1681 tcaagcgcca gttcagctac tacctggtgc agctgtacgc ccccaccacc atgatcgtga
     1741 tcgtgagctg ggtcagcttc tggatcgacc tgcacagcac cgccggccgc gtggccctgg
     1801 gcgtgaccac cctgctgacc atgaccacca tgcagagcgc catcaacgcc aagctgcccc
     1861 ccgtgtccta cgtgaaagtc gtggacgtgt ggctgggcgc ctgccagacc ttcgtgttcg
     1921 gcgccctgct ggagtacgcc ttcgtctcct accaagactc cgtgcgccag aacgaccgct
     1981 cccgcgagaa ggcggcccgc aaggcccaga gacgccgcga gaagctggag atggtggacg
     2041 ccgaggtgta ccagcccccc tgcacgatgg tgagcaaggg cgaggagctg ttcaccgggg
     2101 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2161 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2221 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctggggc gtgcagtgct
     2281 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2341 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2401 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2461 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacatcagc cacaacgtct
     2521 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaaca
     2581 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2641 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2701 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2761 tcggcatgga cgagctgtac aagacgtgcc acaccttcga ggccagagag accttccgcg
     2821 acaaagtcag acgctacttc accaagcccg actacctgcc cgccaagatc gacttctacg
     2881 cccgcttcgt ggtgcccctg gccttcctgg ccttcaacgt gatctactgg gtgtcctgcc
     2941 tgatcatgag cgctaacgcc agcacccccg agtccctggt gtaggctagc aagggcaatt
     3001 ctgcagatat ccagcacagt ggcggccgct cgagtctaga gggcccgcgg ttcgaaggta
     3061 agcctatccc taaccctctc ctcggtctcg attctacgcg taccggtcat catcaccatc
     3121 accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3181 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3241 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3301 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3361 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3421 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3481 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3541 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg catcccttta gggttccgat
     3601 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3661 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3721 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3781 tataagggat tttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3841 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3901 cccaggcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3961 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4021 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4081 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     4141 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     4201 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     4261 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     4321 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     4381 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     4441 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     4501 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     4561 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     4621 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     4681 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     4741 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     4801 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     4861 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     4921 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     4981 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     5041 ttgacgagtt cttctgagcg ggactctggg gttcgcgaaa tgaccgacca agcgacgccc
     5101 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5161 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5221 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5281 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5341 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     5401 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5461 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5521 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5581 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5641 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5701 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5761 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5821 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5881 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5941 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     6001 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6061 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6121 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6181 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6241 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6301 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6361 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6421 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6481 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6541 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6601 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6661 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6721 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6781 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6841 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6901 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6961 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7021 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7081 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7141 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7201 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7261 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7321 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7381 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7441 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7501 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
