LOCUS       sequence-317190-        6373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of human MFSD12 with recoded nucleotide 
            sequence. Dileucine motif LL256-257 has been mutated to AA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6373)
  AUTHORS   Adelmann CH, Traunbauer AK, Chen B, Condon KJ, Chan SH, Kunchok T, 
            Lewis CA, Sabatini DM
  TITLE     MFSD12 mediates the import of cysteine into melanosomes and 
            lysosomes.
  JOURNAL   Nature. 2020 Nov 18. pii: 10.1038/s41586-020-2937-x. doi: 
            10.1038/s41586-020-2937-x.
  PUBMED    33208952
REFERENCE   2  (bases 1 to 6373)
  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: "Nature. 
            2020 Nov 18. pii: 10.1038/s41586-020-2937-x. doi: 
            10.1038/s41586-020-2937-x."
COMMENT     Sequence Label: prk5-MFSD12-TEV-3xFLAG-3xHA PM mutant
FEATURES             Location/Qualifiers
     source          1..6373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      699..1287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1188..1207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1441..1458
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1575..1596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1611..1616,1617..1634,1635..1641)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1649..1665
                     /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)."
     primer_bind     1654..1676
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1673..1689
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1673..1689
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        1718..2097
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2098..2301
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2252..2272
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2298..2322
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2532..2550
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2532..2549
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2563..2582
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      2635..2644
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4090..4110
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4117..4206
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4234..4299
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4330..4356
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      4377..4386
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4390..4524
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4427..4446)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4481..4500
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4593..4950
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4593..4784
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4616..4636)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4801..4936
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4970..4987)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4970..4986)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4979..5001)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5199..5654
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5286..5305)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5496..5517
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5671..5690)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5790..5812
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5850..5868)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5936..6040
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6041..6109,6110..6373,1..528)
                     /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     complement(6259..6278)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
       61 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
      121 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
      181 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
      241 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
      301 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
      361 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
      421 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
      481 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
      541 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
      601 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
      661 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
      721 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
      781 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
      841 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
      901 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
      961 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1021 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1081 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1141 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     1201 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     1261 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     1321 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     1381 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     1441 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     1501 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     1561 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     1621 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     1681 agctatgaca tgattacgaa ttaattcgag ctcgcccgac attgattatt gactagttat
     1741 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     1801 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     1861 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     1921 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     1981 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     2041 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     2101 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     2161 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     2221 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     2281 gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
     2341 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg
     2401 tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa gtaccgccta tagagtctat
     2461 aggcccaccc ccttggcttc gttagaacgc ggctacaatt aatacataac cttatgtatc
     2521 atacacatac gatttaggtg acactataga ataacatcca ctttgccttt ctctccacag
     2581 gtgtccactc ccaggtccaa ctgcacctcg gttctatcga ttgaattcgt cgacgccacc
     2641 atgggtcctg gtccacctgc cgctggcgcg gctccatctc cacggcccct gagtctcgta
     2701 gcccggttgt cctatgccgt gggccatttc cttaatgacc tttgtgccag tatgtggttt
     2761 acctacttgt tgctttatct ccatagcgtg agagcttatt cttctcgagg agcgggtttg
     2821 ctcctgttgc tgggccaagt tgcagatggc ctgtgcaccc ctcttgtggg ttatgaggcc
     2881 gatagagcgg caagttgctg tgcaaggtat ggtccgcgaa aggcatggca tttggttggc
     2941 accgtatgcg tacttctttc tttcccattt atcttctctc catgtctcgg ctgcggcgca
     3001 gccacccccg aatgggctgc tttgttgtat tacggacctt tcatcgtaat attccaattc
     3061 ggctgggcgt caacgcaaat tagccatctc agtctcattc cggaacttgt tactaatgat
     3121 cacgaaaaag tagagcttac cgcgttgcgc tatgccttca ccgttgtagc caacataacg
     3181 gtatacggcg ctgcctggct tctcttgcac ctccagggga gctcaagggt cgagccaacg
     3241 caggatattt caatatcaga ccagctcggt ggtcaggatg ttccggtgtt ccggaatctg
     3301 agtttgctcg tcgtcggagt tggagctgtc ttcagccttc ttttccacct tggaacaagg
     3361 gaacgcagac ggcctcacgc tgaagagcca ggtgagcaca ctccggctgc agcccctgct
     3421 acagcgcaac ccttgcttct ctggaagcat tggctgagag aacccgcttt ctatcaagtg
     3481 ggcatactgt atatgacaac gaggcttatt gtgaatctga gtcaaaccta tatggccatg
     3541 tatttgacat attctcttca cttgcctaaa aagttcatcg ccacaattcc gcttgttatg
     3601 tatctgagtg gtttcttgag tagctttctg atgaagccga tcaacaagtg cattggacgg
     3661 aacatgacgt acttcagtgg ccttttggtc atcctggctt ttgcagcatg ggttgctctt
     3721 gcggagggcc tgggagtagc agtgtatgcg gctgctgttc tgttgggggc cgggtgtgca
     3781 acaatcctcg tgacgtccct tgcgatgacg gcagatctga ttgggcctca cacgaactcc
     3841 ggagccttcg tttacggttc tatgtccttc ttggacaagg ttgctaatgg gcttgccgtg
     3901 atggcaattc aatcccttca cccgtgccct tctgagttgt gctgcagagc gtgtgtgtcc
     3961 ttttatcatt gggctatggt cgctgtgacg ggtggagtag gggtggcagc agccctctgc
     4021 ctctgtagtc ttttgctgtg gccgactagg cttcgccgct gggaccggga cgcccgcccg
     4081 gcggccgcag aaaacctgta cttccaaggt cccgtttacc catacgatgt tcctgactat
     4141 gcgggctatc cctatgacgt cccggactat gcaggatcct atccatatga cgttccagat
     4201 tacgctccgg ccgccggatc cggatccgca ggcgactaca aagaccatga cggtgattat
     4261 aaagatcatg acattgatta caaggatgac gatgacaagt aataataagc ggccgcagga
     4321 ggtggaggtt acccctatga tgtgcctgac tacgcatgat aaggatcccc agcttggccg
     4381 ccatggccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4441 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4501 aatgtatctt atcatgtctg gatcgggaat taattcggcg cagcaccatg gcctgaaata
     4561 acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat
     4621 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4681 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4741 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4801 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4861 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4921 ggcttttttg gaggcctagg cttttgcaaa aagctgttaa cagcttggca ctggccgtcg
     4981 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     5041 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     5101 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     5161 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     5221 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5281 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5341 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5401 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5461 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5521 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     5581 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5641 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5701 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5761 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5821 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5881 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5941 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6001 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     6061 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6121 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6181 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6241 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6301 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6361 agaaaagcat ctt
//
