LOCUS       sequence-324224-        7369 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses STAM1 in mammalian cells with a GFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7369)
  AUTHORS   Bonet-Ponce L, Beilina A, Williamson CD, Lindberg E, Kluss JH, 
            Saez-Atienzar S, Landeck N, Kumaran R, Mamais A, Bleck CKE, Li Y, 
            Cookson MR
  TITLE     LRRK2 mediates tubulation and vesicle sorting from lysosomes.
  JOURNAL   Sci Adv. 2020 Nov 11;6(46). pii: 6/46/eabb2454. doi: 
            10.1126/sciadv.abb2454. Print 2020 Nov.
  PUBMED    33177079
REFERENCE   2  (bases 1 to 7369)
  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 Adv."; 
            date: "2020-11-11"; volume: "6(46). pii: 6/46/eabb2454. doi"; pages:
            " 10.1126/sciadv.abb2454. Print 2020 Nov"
COMMENT     Sequence Label: GFP-STAM1
FEATURES             Location/Qualifiers
     source          1..7369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(905..907,908..910,911..1621)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(938..966)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1561..1581
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    1643..1667
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2904..2928)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2960..2977)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2966..3190
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3236..3664
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3323..3342)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3533..3554
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3673..3693)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3678..4007
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3858..3993
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3920..3939
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4074..4868
                     /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(4128..4147)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4738..4757
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5042..5163
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5079..5098)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5133..5152
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5212..5228)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5212..5228)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5225..5247)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5236..5252
                     /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(5260..5266,5267..5284,5285..5290))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5305..5326
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5443..5460)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5614..6202)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5694..5713)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6373..7164,7165..7233))
                     /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     6996..7015
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7234..7338)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
      901 caccatggcc agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
      961 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgctac
     1021 atacggaaag cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc
     1081 aacacttgtc actactttct cttatggtgt tcaatgcttt tcccgttatc cggatcatat
     1141 gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat
     1201 atctttcaaa gatgacggga actacaagac gcgtgctgaa gtcaagtttg aaggtgatac
     1261 ccttgttaat cgtatcgagt taaaaggtat tgattttaaa gaagatggaa acattctcgg
     1321 acacaaactc gagtacaact ataactcaca caatgtatac atcacggcag acaaacaaaa
     1381 gaatggaatc aaagctaact tcaaaattcg ccacaacatt gaagatggat ccgttcaact
     1441 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa
     1501 ccattacctg tcgacacaat ctgccctttc gaaagatccc aacgaaaagc gtgaccacat
     1561 ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa
     1621 aagcggttcc ggaccggatc aaacaagttt gtacaaaaaa gcaggctccg aattcgccct
     1681 tcctcttttt gccaccaatc ccttcgatca ggatgttgag aaagcaacca gcgagatgaa
     1741 tactgctgag gactggggcc tcattttgga tatctgtgat aaagttggtc agtctcgcac
     1801 tggacctaag gattgtcttc ggtctattat gagaagagtg aaccacaaag atcctcacgt
     1861 tgctatgcag gctttgactc ttctaggagc atgtgtatca aactgtggca aaatttttca
     1921 tttagaagta tgttcaagag attttgctag tgaagtaagc aacgtattaa ataagggtca
     1981 tcctaaagta tgtgaaaaat taaaggctct tatggttgaa tggacagatg aatttaagaa
     2041 tgatccacag cttagtctaa tatcagcaat gattaagaac cttaaggaac aaggagttac
     2101 gttcccagct attggctctc aggctgcaga acaagcaaaa gcaagcccag ctcttgtagc
     2161 caaggatcct ggtactgtgg ctaacaaaaa agaagaagaa gatttagcaa aagccattga
     2221 gttgtctctc aaggaacaaa ggcagcagtc aaccaccctt tccactttgt atccaagcac
     2281 atccagtctc ttaactaacc accaacatga aggccgaaaa gttcgtgcta tatatgactt
     2341 tgaagctgct gaagacaatg aacttacttt taaagctgga gaaattatta cagttcttga
     2401 tgacagtgat cctaactggt ggaaaggtga aacccatcaa ggcatagggt tatttccttc
     2461 taattttgtg actgcatatc tcactgctga accagaaatg attaaaacag agaagaagac
     2521 ggtacaattt agtgatgatg ttcaggtaga gacaatagaa ccagagccgg aaccagcctt
     2581 tattgatgaa gataaaatgg accagttgct acagatgctg caaagtacag accccagtga
     2641 tgatcagcca gacctaccag agctgcttca tcttgaagca atgtgtcacc agatgggacc
     2701 tctcattgat gaaaagctgg aagatattga tagaaaacat tcagaactct cagaacttaa
     2761 tgtgaaagtg atggaggccc tttccttata taccaagtta atgaacgaag atccgatgta
     2821 ttccatgtat gcaaagttac agaatcagcc aggcagtggt cccaccatcc gcaaacccag
     2881 cccttcctag aagggcgaat tcgacccagc tttcttgtac aaagtggtga taattaatta
     2941 agataaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3001 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3061 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3121 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3181 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3241 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3301 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3361 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3421 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3481 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3541 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3601 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3661 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3721 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3781 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3841 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3901 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3961 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     4021 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     4081 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4141 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4201 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4261 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4321 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4381 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4441 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4501 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4561 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4621 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4681 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4741 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4801 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4861 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4921 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4981 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5041 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5101 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5161 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5221 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5281 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5341 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5401 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5461 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5521 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5581 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5641 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5701 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5761 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5821 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5881 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5941 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6001 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6061 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6121 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6181 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6241 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6301 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6361 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6421 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6481 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6541 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6601 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6661 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6721 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6781 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6841 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6901 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6961 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7021 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7081 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7141 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7201 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7261 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7321 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
