LOCUS       sequence-378582-        7778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for human CBS mutant H65R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  AUTHORS   Switzer CH, Kasamatsu S, Ihara H, Eaton P
  TITLE     SOD1 is an essential H(2)S detoxifying enzyme.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jan 17;120(3):e2205044120. doi: 
            10.1073/pnas.2205044120. Epub 2023 Jan 11.
  PUBMED    36630448
REFERENCE   2  (bases 1 to 7778)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-01-17"; volume: "120(3):e2205044120. 
            doi"; pages: " 10.1073/pnas.2205044120. Epub 2023 Jan 11"
COMMENT     Sequence Label: GFP-CBS (H65R)
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     782..801
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        782..800
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             842..1555
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(842..844,845..1555)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(884..905)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1145..1164)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1492..1513
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3288..3305)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3294..3518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3564..3992
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3651..3670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3861..3882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4001..4021)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4006..4335
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4186..4321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4248..4267
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4402..5196
                     /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(4456..4475)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5066..5085
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5370..5491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5407..5426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5461..5480
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5540..5556)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5540..5556)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5553..5575)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5564..5580
                     /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(5588..5594,5595..5612,5613..5618))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5633..5654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5771..5788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5942..6530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6022..6041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6701..7492,7493..7561))
                     /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     7324..7343
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7562..7666)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7741..7760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
       61 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
      121 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa
      781 ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaactta agcttgccac
      841 catgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      901 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      961 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1021 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1081 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1141 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1201 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1261 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1321 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1381 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1441 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1501 gctggagttc gtgaccgccg ccgggatcac tcacggcatg gacgagctgt acaagggtac
     1561 catgccttct gagacccccc aggcagaagt ggggcccaca ggctgccccc accgctcagg
     1621 gccacactcg gcgaagggga gcctggagaa ggggtcccca gaggataagg aagccaagga
     1681 gcccctgtgg atccggcccg atgctccgag caggtgcacc tggcagctgg gccggcctgc
     1741 ctccgagtcc ccacgtcacc acactgcccc ggcaaaatct ccaaaaatct tgccagatat
     1801 tctgaagaaa atcggggaca cccctatggt cagaatcaac aagattggga agaagttcgg
     1861 cctgaagtgt gagctcttgg ccaagtgtga gttcttcaac gcgggcggga gcgtgaagga
     1921 ccgcatcagc ctgcggatga ttgaggatgc tgagcgcgac gggacgctga agcccgggga
     1981 cacgattatc gagccgacat ccgggaacac cgggatcggg ctggccctgg ctgcggcagt
     2041 gaggggctat cgctgcatca tcgtgatgcc agagaagatg agctccgaga aggtggacgt
     2101 gctgcgggca ctgggggctg agattgtgag gacgcccacc aatgccaggt tcgactcccc
     2161 ggagtcacac gtgggggtgg cctggcggct gaagaacgaa atccccaatt ctcacatcct
     2221 agaccagtac cgcaacgcca gcaaccccct ggctcactac gacaccaccg ctgatgagat
     2281 cctgcagcag tgtgatggga agctggacat gctggtggct tcagtgggca cgggcggcac
     2341 catcacgggc attgccagga agctgaagga gaagtgtcct ggatgcagga tcattggggt
     2401 ggatcccgaa gggtccatcc tcgcagagcc ggaggagctg aaccagacgg agcagacaac
     2461 ctacgaggtg gaagggatcg gctacgactt catccccacg gtgctggaca ggacggtggt
     2521 ggacaagtgg ttcaagagca acgatgagga ggcgttcacc tttgcccgca tgctgatcgc
     2581 gcaagagggg ctgctgtgcg gtggcagtgc tggcagcacg gtggcggtgg ccgtgaaggc
     2641 cgcgcaggag ctgcaggagg gccagcgctg cgtggtcatt ctgcccgact cagtgcggaa
     2701 ctacatgacc aagttcctga gcgacaggtg gatgctgcag aagggctttc tgaaggagga
     2761 ggacctcacg gagaagaagc cctggtggtg gcacctccgt gttcaggagc tgggcctgtc
     2821 agccccgctg accgtgctcc cgaccatcac ctgtgggcac accatcgaga tcctccggga
     2881 gaagggcttc gaccaggcgc ccgtggtgga tgaggcgggg gtaatcctgg gaatggtgac
     2941 gcttgggaac atgctctcgt ccctgcttgc cgggaaggtg cagccgtcag accaagttgg
     3001 caaagtcatc tacaagcagt tcaaacagat ccgcctcacg gacacgctgg gcaggctctc
     3061 gcacatcctg gagatggacc acttcgccct ggtggtgcac gagcagatcc agtaccacag
     3121 caccgggaag tccagtcagc ggcagatggt gttcggggtg gtcaccgcca ttgacttgct
     3181 gaacttcgtg gccgcccagg agcgggacca gaagtgagaa ttctgcagat atccagcaca
     3241 gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3301 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3361 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3421 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3481 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3541 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3601 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3661 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3721 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3781 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3841 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3901 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3961 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     4021 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4081 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4141 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4201 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4261 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4321 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4381 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4441 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4501 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4561 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4621 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4681 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4741 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4801 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4861 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4921 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4981 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5041 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5101 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5161 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5221 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5281 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5341 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5401 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5461 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5521 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5581 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5641 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5701 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5761 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5821 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5881 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5941 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6001 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6061 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6121 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6181 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6241 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6301 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6361 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6421 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6481 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6541 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6601 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6661 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6721 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6781 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6841 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6901 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6961 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7021 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7081 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7141 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7201 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7261 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7321 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7381 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7441 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7501 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7561 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7621 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7681 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     7741 tacaatctgc tctgatgccg catagttaag ccagtatc
//
