LOCUS       sequence-178488-        6589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses YFP-DAT R60A mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6589)
  AUTHORS   Ma S, Cheng MH, Guthrie DA, Newman AH, Bahar I, Sorkin A
  TITLE     Targeting of dopamine transporter to filopodia requires an 
            outward-facing conformation of the transporter.
  JOURNAL   Sci Rep. 2017 Jul 14;7(1):5399. doi: 10.1038/s41598-017-05637-x.
  PUBMED    28710426
REFERENCE   2  (bases 1 to 6589)
  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: "Sci Rep."; 
            date: "2017-07-14"; volume: "7(1):5399. doi"; pages: " 
            10.1038/s41598-017-05637-x"
COMMENT     Sequence Label: YFP-DAT R60A
FEATURES             Location/Qualifiers
     source          1..6589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1813..1824
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     polyA_signal    3377..3498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3414..3433)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3468..3487
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3505..3960)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3642..3663)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3854..3873
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3987..4091
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4093..4450
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4093..4284
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4116..4136)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4301..4436
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4485..5279
                     /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             4485..5279
                     /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(4539..4558)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5149..5168
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5467..5486)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5511..5558
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5887..6475
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6376..6395
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc
     1381 atgagtaaga gcaaatgctc cgtgggactc atgtcttccg tggtggcccc ggctaaggag
     1441 cccaatgccg tgggcccgaa ggaggtggag ctcatccttg tcaaggagca gaacggagtg
     1501 cagctcacca gctccaccct caccaacccg cggcagagcc ccgtggaggc ccaggatgcg
     1561 gagacctggg gcaagaagat cgactttctc ctgtccgtca ttggctttgc tgtggacctg
     1621 gccaacgtct ggcggttccc ctacctgtgc tacaaaaatg gtggcggtgc cttcctggtc
     1681 ccctacctgc tcttcatggt cattgctggg atgccacttt tctacatgga gctggccctc
     1741 ggccagttca acagggaagg ggccgctggt gtctggaaga tctgccccat actgaaaggt
     1801 gtgggcttca cggtcatcct catctcactg tatgtcggct tcttctacaa cgtcatcatc
     1861 gcctgggcgc tgcactatct cttctcctcc ttcaccacgg agctcccctg gatccactgc
     1921 aacaactcct ggaacagccc caactgctcg gatgcccatc ctggtgactc cagtggagac
     1981 agctcgggcc tcaacgacac ttttgggacc acacctgctg ccgagtactt tgaacgtggc
     2041 gtgctgcacc tccaccagag ccatggcatc gacgacctgg ggcctccgcg gtggcagctc
     2101 acagcctgcc tggtgctggt catcgtgctg ctctacttca gcctctggaa gggcgtgaag
     2161 acctcaggga aggtggtatg gatcacagcc accatgccat acgtggtcct cactgccctg
     2221 ctcctgcgtg gggtcaccct ccctggagcc atagacggca tcagagcata cctgagcgtt
     2281 gacttctacc ggctctgcga ggcgtctgtt tggattgacg cggccaccca ggtgtgcttc
     2341 tccctgggcg tggggttcgg ggtgctgatc gccttctcca gctacaacaa gttcaccaac
     2401 aactgctaca gggacgcgat tgtcaccacc tccatcaact ccctgacgag cttctcctcc
     2461 ggcttcgtcg tcttctcctt cctggggtac atggcacaga agcacagtgt gcccatcggg
     2521 gacgtggcca aggacgggcc agggctgatc ttcatcatct acccggaagc catcgccacg
     2581 ctccctctgt cctcagcctg ggccgtggtc ttcttcatca tgctgctcac cctgggtatc
     2641 gacagcgcca tgggtggtat ggagtcagtg atcaccgggc tcatcgatga gttccagctg
     2701 ctgcacagac accgtgagct cttcacgctc ttcatcgtcc tagcgacctt cctcctgtcc
     2761 ctgttctgcg tcaccaacgg tggcatctac gtcttcacgc tcctggacca ttttgcagcc
     2821 ggcacgtcca tcctctttgg agtgctcatc gaagccatcg gagtggcctg gttctatggt
     2881 gttgggcagt tcagcgacga catccagcag atgaccgggc agcggcccag cctgtactgg
     2941 cggctgtgct ggaagctggt cagcccctgc tttctcctgt tcgtggtcgt ggtcagcatt
     3001 gtgaccttca gaccccccca ctacggagcc tacatcttcc ccgactgggc caacgcgctg
     3061 ggctgggtca tcgccacatc ctccatggcc atggtgccca tctatgcggc ctacaagttc
     3121 tgcagcctgc ctgggtcctt tcgagagaaa ctggcctacg ccattgcacc cgagaaggac
     3181 cgtgagctgg tggacagagg ggaggtgcgc cagttcacgc tccgccactg gctcaaggtg
     3241 tagcccggga tccaccggat ctagataact gatcataatc agccatacca catttgtaga
     3301 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     3361 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3421 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3481 actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     3541 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3601 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3661 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3721 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3781 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3841 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3901 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3961 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4021 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4081 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     4141 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4201 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4261 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4321 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4381 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4441 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4501 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4561 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4621 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     4681 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4741 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4801 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4861 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4921 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4981 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     5041 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     5101 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     5161 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     5221 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     5281 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     5341 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5401 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     5461 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     5521 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     5581 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     5641 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     5701 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     5761 acttcatttt taatttaaaa gaatctaggt gaagatcctt tttgataatc tcatgaccaa
     5821 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5881 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5941 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6001 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6061 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6121 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6181 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6241 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6301 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6361 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6421 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6481 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6541 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
