LOCUS       sequence-316510-        8365 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of human Rap2a..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8365)
  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 8365)
  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: pCHA1.1-Rap2a-V5
FEATURES             Location/Qualifiers
     source          1..8365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        1824..3035
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     2872..2891
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3610..3651
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    3696..3813
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3862..4372
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4291..4309
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4392..4787
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             4884..5117
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5189..5310
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5226..5245)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5280..5299
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5350..5485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5505..5524)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5506..5524)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5534..5551)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5534..5550)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5543..5565)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5692..6147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5779..5798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5989..6010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6173..6277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6278..6346,6347..7138)
                     /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(6496..6515)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7309..7897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7798..7817
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8051..8068
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8185..8206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8221..8226,8227..8244,8245..8251)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8259..8275
                     /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     8264..8286
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8283..8299
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8283..8299
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8318..8338
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8320..8338
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcag cagctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
     1801 gagactagcc tcgagcggga tctggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc
     1861 ccccctcctc acggcgagcg ctgccacgtc agacgaaggg cgcagcgagc gtcctgatcc
     1921 ttccgcccgg acgctcagga cagcggcccg ctgctcataa gactcggcct tagaacccca
     1981 gtatcagcag aaggacattt taggacggga cttgggtgac tctagggcac tggttttctt
     2041 tccagagagc ggaacaggcg aggaaaagta gtcccttctc ggcgattctg cggagggatc
     2101 tccgtggggc ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg cacagctagt
     2161 tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt
     2221 ggtgagtagc gggctgctgg gctggccggg gctttcgtgg ccgccgggcc gctcggtggg
     2281 acggaagcgt gtggagagac cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc
     2341 tgaactgggg gttgggggga gcgcagcaaa atggcggctg ttcccgagtc ttgaatggaa
     2401 gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt ggggggcatg gtgggcggca
     2461 agaacccaag gtcttgaggc cttcgctaat gcgggaaagc tcttattcgg gtgagatggg
     2521 ctggggcacc atctggggac cctgacgtga agtttgtcac tgactggaga actcggtttg
     2581 tcgtctgttg cgggggcggc agttatggcg gtgccgttgg gcagtgcacc cgtacctttg
     2641 ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct gttggcttat aatgcagggt
     2701 ggggccacct gccggtaggt gtgcggtagg cttttctccg tcgcaggacg cagggttcgg
     2761 gcctagggta ggctctcctg aatcgacagg cgccggacct ctggtgaggg gagggataag
     2821 tgaggcgtca gtttctttgg tcggttttat gtacctatct tcttaagtag ctgaagctcc
     2881 ggttttgaac tatgcgctcg gggttggcga gtgtgttttg tgaagttttt taggcacctt
     2941 ttgaaatgta atcatttggg tcaatatgta attttcagtg ttagactagt aaattgtccg
     3001 ctaaattctg gccgtttttg gcttttttgt tagacgaagc gctagcccac catgcgcgag
     3061 tacaaagtgg tggtgctggg ctcgggcggg gtaggcaaat ccgccctgac cgtgcagttc
     3121 gtgaccggca ccttcatcga gaaatacgac cccaccatcg aggacttcta ccgcaaggag
     3181 atcgaggtgg attcgtcgcc gtcggtgctg gagatcctgg acacggcggg caccgagcag
     3241 ttcgcgtcca tgcgggacct gtacatcaag aacggccagg gcttcatcct cgtctacagc
     3301 ctcgtcaacc agcagagctt ccaggacatc aagcccatgc gggaccagat catccgcgtg
     3361 aagcggtatg agaaagtgcc agtcatcttg gttgggaaca aagtggacct ggaaagtgag
     3421 agagaagtat cgtccagcga aggcagagcc cttgctgaag agtggggctg cccctttatg
     3481 gaaacttccg ctaagagtaa aacaatggtg gacgaactct ttgcagaaat tgtgaggcag
     3541 atgaactatg ctgctcagcc tgacaaagat gacccatgct gttctgcatg taacatacaa
     3601 gcggccgccg gtaagcctat ccctaaccct ctcctcggtc tcgattctac gtaagaattc
     3661 tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     3721 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     3781 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     3841 ccactttggc cgcggctcga gggggttggg gttgcgcctt ttccaaggca gccctgggtt
     3901 tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg
     3961 gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt
     4021 gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc
     4081 ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca
     4141 gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc
     4201 agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc
     4261 ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg
     4321 cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agggggatct
     4381 gatccaccac catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
     4441 cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc gcagctctct
     4501 ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg
     4561 cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg
     4621 tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc
     4681 ttctcgatct gcatcctggg atcaaagcca tagtgaagga cagtgatgga cagccgacgg
     4741 cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggcctg cagctgcagt
     4801 agtaaatgca tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     4861 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     4921 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     4981 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     5041 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     5101 gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc
     5161 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa
     5221 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5281 tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta
     5341 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     5401 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     5461 tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt
     5521 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     5581 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     5641 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     5701 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5761 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5821 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5881 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5941 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     6001 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     6061 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     6121 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     6181 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6241 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6301 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6361 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6421 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6481 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6541 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6601 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6661 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6721 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6781 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6841 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6901 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6961 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     7021 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     7081 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     7141 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7201 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7261 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7321 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7381 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7441 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7501 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7561 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7621 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7681 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7741 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7801 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7861 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7921 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7981 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     8041 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     8101 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     8161 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     8221 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     8281 cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga
     8341 acaaaagctg gagctgcaag cttaa
//
