LOCUS       sequence-8218-ex        7644 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 7644)
  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 7644)
  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 alpha EYFP
FEATURES             Location/Qualifiers
     source          1..7644
                     /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(2199..2201,2202..2204,2205..2915)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2244..2265)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2505..2524)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2852..2873
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3141..3182
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3192..3209
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3232..3249)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3238..3462
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3508..3936
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3595..3614)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3805..3826
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3945..3965)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3950..4280
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4131..4266
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4193..4212
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4347..5141
                     /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(4401..4420)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5011..5030
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5317..5438
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5354..5373)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5408..5427
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5487..5503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5487..5503)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5500..5522)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5511..5527
                     /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(5535..5541,5542..5559,5560..5565))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5580..5601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5718..5735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5889..6477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5969..5988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6648..7439,7440..7508))
                     /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     7271..7290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7509..7613)
                     /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 tggccacgtg gatcgtgggc aagctgatca tcgcttccct gatcctgggg atccaggccc
     1021 agcaggcccg caccaagagc caggacatct tcgaggacga caacgacaac ggcactacca
     1081 ccctggagtc cctggctcgc ctgacctccc ccatccacat ccccatcgag cagccccaga
     1141 cctccgacag caagatcctg gcccacctgt tcaccagcgg ctacgacttc cgcgtgcgcc
     1201 cccccaccga caacggcggc cccgtggtgg tgagcgtgaa catgctgctg cgcaccatca
     1261 gcaagatcga cgtggtgaac atggagtact ccgcccagct gaccctgcgc gagagctgga
     1321 tcgacaagcg cctgtcctac ggcgtcaagg gcgacggcca gcccgacttc gtcatcctga
     1381 ccgtgggcca ccagatctgg atgcccgaca ccttcttccc caacgagaag caggcttaca
     1441 agcacaccat cgacaagccc aacgtcctga tccgcatcca caacgacggc accgtcctgt
     1501 actccgtccg catcagcctg gtgctgagct gccccatgta cctgcagtac tacccgatgg
     1561 acgtgcagca gtgcagcatc gacctggcct cctacgccta caccaccaag gacatcgagt
     1621 atctgtggaa ggagcacagc cccctgcagc tgaaggtcgg cctgtcctcc agcctgccca
     1681 gcttccagct gaccaacacc agcaccacct actgcaccag cgtcaccaac accggcatct
     1741 acagctgcct gaggaccacc atccagctga agcgcgagtt ctccttctac ctgctgcaac
     1801 tgtacatccc cagctgcatg ctcgtgatcg tgtcctgggt gtccttctgg ttcgaccgca
     1861 ccgccatccc cgcccgcgtc accctgggcg tgaccaccct gctgaccatg accgctcagt
     1921 ccgctggcat caacagccag ctgccccccg tgagctacat caaggccatc gacgtgtgga
     1981 tcggcgcctg catgaccttc atcttctgcg ccctgctgga gttcgccctg gttaaccaca
     2041 tcgcgaacaa gcagggcgtg gagcgcaagg cccgcaccga gcgcgagaag gccgagatcc
     2101 ccctgctgca gaacctgcac aacgacgtcc ccaccaaggt gttcaaccag gaggagaaag
     2161 tccgcaccgt gcccctgaac cgccggcaga tgaattccat ggtgagcaag ggcgaggagc
     2221 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2281 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2341 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg
     2401 gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2461 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2521 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2581 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2641 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2701 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2761 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc
     2821 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2881 ccgggatcac tctcggcatg gacgagctgt acaagaattc cttcctgaac ctgctggaga
     2941 ccaagaccga gtggaacgac atcagcaagc gcgtggacct gatcagccgc gccctgttcc
     3001 ccgtgctgtt cttcgtgttc aacatcctgt actggagccg cttcggccag cagaacgttc
     3061 tgttctgagc tagcaagggc aattctgcag atatccagca cagtggcggc cgctcgagtc
     3121 tagagggccc gcggttcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta
     3181 cgcgtaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     3241 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3301 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3361 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3421 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3481 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3541 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3601 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3661 ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3721 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3781 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3841 ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa
     3901 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3961 agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca
     4021 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4081 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4141 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4201 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4261 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     4321 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     4381 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     4441 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     4501 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     4561 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4621 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4681 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4741 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4801 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4861 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4921 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4981 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     5041 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5101 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcgc
     5161 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     5221 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5281 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     5341 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5401 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     5461 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5521 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5581 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5641 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5701 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5761 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5821 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5881 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5941 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6001 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6061 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     6121 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6181 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6241 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6301 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     6361 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6421 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6601 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6661 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6721 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6781 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6841 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6901 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7621 ccccgaaaag tgccacctga cgtc
//
