LOCUS       sequence-433004-        6423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 10xHis-SUMO (Smt3) fusion to AP3M1 
            amphipathic helix (135-151) fused to mGreenLantern.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6423)
  AUTHORS   Begley M, Aragon M, Baker RW
  TITLE     A structure-based mechanism for initiation of AP-3 coated vesicle 
            formation.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Dec 24;121(52):e2411974121. doi: 
            10.1073/pnas.2411974121. Epub 2024 Dec 20.
  PUBMED    39705307
REFERENCE   2  (bases 1 to 6423)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-12-24"; volume: "121(52):e2411974121. 
            doi"; pages: " 10.1073/pnas.2411974121. Epub 2024 Dec 20"
COMMENT     Sequence Label: MZB208
FEATURES             Location/Qualifiers
     source          1..6423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             303..332
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(687..689,690..692,693..1406)
                     /codon_start=1
                     /product="monomeric GFP derivative that displays 
                     exceptionally high brightness in cells (Campbell et al., 
                     2020)"
                     /label=mGreenLantern
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIVLKGIDFKEDGNILGHKLEYNFNSHKVYITADKQKNGIK
                     ANFKTRHNVEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLK
                     ERVTAAGITHDMDELYK"
     primer_bind     complement(732..753)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1496..1513
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1566..1584)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1580..1627
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1664..2119
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1751..1770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1961..1982
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2211..3026)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2211..3026)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2934..2953
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3018..3037
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3148..3736
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3637..3656
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3890..3907
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3922..4061
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3987..4006)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4147..4169
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4163..4354)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5163..6245)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6207..6226
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6246..6323)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgg
      301 gccatcacca tcaccatcac catcaccatc acggcagcat gagcgatagc gaagtgaacc
      361 aggaagcgaa accggaagtg aaaccggaag tgaaaccgga aacccatatt aacctgaaag
      421 tgagcgatgg cagcagcgaa atttttttta aaattaaaaa aaccaccccg ctgcgtcgtc
      481 tgatggaagc gtttgcgaaa cgtcagggca aagaaatgga tagcctgcgt tttctgtatg
      541 atggcattcg tattcaggcg gatcagaccc cggaagatct ggatatggaa gataacgata
      601 ttattgaagc gcatcgtgaa cagattggcg gcaaaccgcc gaccattctg cgcagcgtgg
      661 tgaacagcat taccggcagc agcggcatgg tgagcaaggg cgaggagctg ttcaccgggg
      721 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg
      781 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
      841 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt aggctacggc gtggcctgct
      901 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      961 gctacgtcca ggagcgcacc atctctttca aggacgacgg tacctacaag acccgcgccg
     1021 aggtgaagtt cgagggcgac accctggtga accgcatcgt gctgaagggc atcgacttca
     1081 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaaggtct
     1141 atatcacggc cgacaagcag aagaacggca tcaaggctaa cttcaagacc cgccacaacg
     1201 ttgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1261 gccccgtgct gctgcccgac aaccactacc tgagccatca gtccaagctg agcaaagacc
     1321 ccaacgagaa gcgcgatcac atggtcctga aggagagggt gaccgccgcc gggattacac
     1381 atgacatgga cgagctgtac aagtaataag gatccatggg taccctcgag tgaagacaag
     1441 cttaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttag tcgagcacca
     1501 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1561 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1621 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1681 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1741 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1801 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1861 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1921 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1981 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2041 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2101 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2161 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     2221 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     2281 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     2341 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2401 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2461 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     2521 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     2581 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     2641 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2701 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     2761 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     2821 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     2881 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     2941 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     3001 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     3061 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3121 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3181 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3241 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3301 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3361 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3421 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3481 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3541 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3601 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3661 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3721 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3781 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3841 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3901 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3961 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4021 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     4081 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4141 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4201 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     4261 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4321 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4381 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4441 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4501 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4561 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4621 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4681 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4741 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4801 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4861 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4921 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4981 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     5041 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5101 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     5161 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5221 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     5281 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5341 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5401 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5461 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5521 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5581 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5641 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5701 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5761 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5821 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5881 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5941 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     6001 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     6061 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     6121 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     6181 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     6241 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6301 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6361 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6421 atg
//
