LOCUS       sequence-176110-        7183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RFP and M1C448Y spastin in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7183)
  AUTHORS   Solowska JM, D'Rozario M, Jean DC, Davidson MW, Marenda DR, Baas PW
  TITLE     Pathogenic mutation of spastin has gain-of-function effects on 
            microtubule dynamics.
  JOURNAL   J Neurosci. 2014 Jan 29;34(5):1856-67. doi: 
            10.1523/JNEUROSCI.3309-13.2014.
  PUBMED    24478365
REFERENCE   2  (bases 1 to 7183)
  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: 
            "10.1523/JNEUROSCI.3309-13"; journalName: "J Neurosci"; date: 
            "2014-01-29- 29"; volume: "34"; issue: "5"; pages: "1856-67"
COMMENT     Sequence Label: pIRES RED/SP M1C448Y
FEATURES             Location/Qualifiers
     source          1..7183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    2467..2523
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    join(2525..3099,3100..3102,3103..3111)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2692..2709)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2919..2938
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3112..3789
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(3292..3312)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3510..3529
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3704..3727
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3911..4032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3948..3967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4002..4021
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4039..4494)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4176..4197)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4388..4407
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4521..4625
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4627..4984
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4627..4818
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4650..4670)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4835..4970
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5019..5813
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5019..5813
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5073..5092)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5683..5702
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6001..6020)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6045..6092
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6421..7009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6910..6929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatcg
      541 cggcggcggc agtgagaggc caccatgaat tctccgggtg gacgagggaa gaagaaaggc
      601 tccggcggcg ccagcaaccc ggtgcctccc aggcctccgc ccccttgcct ggcccccgcc
      661 cctcccgccg ccgggccggc ccctccgccc gagtcgccgc ataagcggaa cctgtactat
      721 ttctcctacc cgctgtttgt aggcttcgcg ctgctgcgtt tggtcgcctt ccacctgggg
      781 ctcctcttcg tgtggctctg ccagcgcttc tcccgcgccc tcatggcagc caagaggagc
      841 tccggggccg cgccagcacc tgcctcggcc tcggccccgg cgccggtgcc gggcggcgag
      901 gccgagcgcg tccgagtctt ccacaaacag gccttcgagt acatctccat tgccctgcgc
      961 atcgatgagg atgagaaagc aggacagaag gagcaagctg tggaatggta taagaaaggt
     1021 attgaagaac tggaaaaagg aatagctgtt atagttacag gacaaggtga acagtgtgaa
     1081 agagctagac gccttcaagc taaaatgatg actaatttgg ttatggccaa ggaccgctta
     1141 caacttctag agaagatgca accagttttg ccattttcca agtcacaaac ggacgtctat
     1201 aatgacagta ctaacttggc atgccgcaat ggacatctcc agtcagaaag tggagctgtt
     1261 ccaaaaagaa aagacccctt aacacacact agtaattcac tgcctcgttc aaaaacagtt
     1321 atgaaaactg gatctgcagg cctttcaggc caccatagag cacctagtta cagtggttta
     1381 tccatggttt ctggagtgaa acagggatct ggtcctgctc ctaccactca taagggtact
     1441 ccgaaaacaa ataggacaaa taaaccttct acccctacaa ctgctactcg taagaaaaaa
     1501 gacttgaaga attttaggaa tgtggacagc aaccttgcta accttataat gaatgaaatt
     1561 gtggacaatg gaacagctgt taaatttgat gatatagctg gtcaagactt ggcaaaacaa
     1621 gcattgcaag aaattgttat tcttccttct ctgaggcctg agttgttcac agggcttaga
     1681 gctcctgcca gagggctgtt actctttggt ccacctggga atgggaagac aatgctggct
     1741 aaagcagtag ctgcagaatc gaatgcaacc ttctttaata taagtgctgc aagtttaact
     1801 tcaaaatacg tgggagaagg agagaaattg gtgagggctc tttttgctgt ggctcgagaa
     1861 cttcaacctt ctataatttt tatagatgaa gttgatagcc ttttgtatga aagaagagaa
     1921 ggggagcacg atgctagtag acgcctaaaa actgaatttc taatagaatt tgatggtgta
     1981 cagtctgctg gagatgacag agtacttgta atgggtgcaa ctaataggcc acaagagctt
     2041 gatgaggctg ttctcaggcg tttcatcaaa cgggtatatg tgtctttacc aaatgaggag
     2101 acaagactac ttttgcttaa aaatctgtta tgtaaacaag gaagtccatt gacccaaaaa
     2161 gaactagcac aacttgctag aatgactgat ggatactcag gaagtgacct aacagctttg
     2221 gcaaaagatg cagcactggg tcctatccga gaactaaaac cagaacaggt gaagaatatg
     2281 tctgccagtg agatgagaaa tattcgatta tctgacttca ctgaatcctt gaaaaaaata
     2341 aaacgcagcg tcagccctca aactttagaa gcgtacatac gttggaacaa ggactttgga
     2401 gataccactg tttaaggaaa tacctttgta aacctgcaga acattttact taaaagagga
     2461 aacacaagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga
     2521 tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     2581 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2641 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     2701 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     2761 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     2821 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     2881 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     2941 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     3001 ggtacacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     3061 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catggatagc
     3121 actgagaacg tcatcaagcc cttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     3181 ggccacgagt tcgagatcga gggcgagggc gagggcaagc cctacgaggg cacccagacc
     3241 gccaagctgc aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc
     3301 cagttccagt acggctccaa ggtgtacgtg aagcaccccg ccgacatccc cgactacaag
     3361 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     3421 gtggtgaccg tgacccagga ctcctccctg caggacggca ccttcatcta ccacgtgaag
     3481 ttcatcggcg tgaacttccc ctccgacggc cccgtaatgc agaagaagac tctgggctgg
     3541 gagccctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccacaag
     3601 gcgctgaagc tgaagggcgg cggccactac ctggtggagt tcaagtcaat ctacatggcc
     3661 aagaagcccg tgaagctgcc cggctactac tacgtggact ccaagctgga catcacctcc
     3721 cacaacgagg actacaccgt ggtggagcag tacgagcgcg ccgaggcccg ccaccacctg
     3781 ttccagtagg cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     3841 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     3901 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3961 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4021 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     4081 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     4141 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     4201 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     4261 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     4321 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     4381 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     4441 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     4501 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4561 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4621 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     4681 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4741 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4801 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4861 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4921 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4981 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     5041 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     5101 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     5161 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     5221 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     5281 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     5341 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     5401 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     5461 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     5521 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     5581 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     5641 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     5701 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     5761 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     5821 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     5881 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5941 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     6001 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     6061 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     6121 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     6181 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     6241 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     6301 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6361 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6421 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6481 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6541 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6601 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6661 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6721 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6781 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6841 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6901 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6961 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7021 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     7081 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     7141 caattacggg gtcattagtt catagcccat atatggagtt ccg
//
