LOCUS       sequence-433002-        6417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 10xHis-SUMO (Smt3) fusion to AP3D1 
            amphipathic helix (2-15) fused to mGreenLantern.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6417)
  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 6417)
  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: MZB207
FEATURES             Location/Qualifiers
     source          1..6417
                     /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(681..683,684..686,687..1400)
                     /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(726..747)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1490..1507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1560..1578)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1574..1621
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1658..2113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1745..1764)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1955..1976
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2205..3020)
                     /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(2205..3020)
                     /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     2928..2947
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3012..3031
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3142..3730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3631..3650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3884..3901
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3916..4055
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3981..4000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4141..4163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4157..4348)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5157..6239)
                     /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     6201..6220
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6240..6317)
                     /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 gcgcgctgaa aatggtgaaa ggcagcattg
      661 atcgcatgtt tgataaaggc atggtgagca agggcgagga gctgttcacc ggggtggtgc
      721 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg
      781 gcgagggcga tgccaccaac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      841 tgcccgtgcc ctggcccacc ctcgtgacca ccttaggcta cggcgtggcc tgcttcgccc
      901 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
      961 tccaggagcg caccatctct ttcaaggacg acggtaccta caagacccgc gccgaggtga
     1021 agttcgaggg cgacaccctg gtgaaccgca tcgtgctgaa gggcatcgac ttcaaggagg
     1081 acggcaacat cctggggcac aagctggagt acaacttcaa cagccacaag gtctatatca
     1141 cggccgacaa gcagaagaac ggcatcaagg ctaacttcaa gacccgccac aacgttgagg
     1201 acggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1261 tgctgctgcc cgacaaccac tacctgagcc atcagtccaa gctgagcaaa gaccccaacg
     1321 agaagcgcga tcacatggtc ctgaaggaga gggtgaccgc cgccgggatt acacatgaca
     1381 tggacgagct gtacaagtaa taaggatcca tgggtaccct cgagtgaaga caagcttagg
     1441 tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttagtcgagc accaccacca
     1501 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     1561 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1621 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     1681 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     1741 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1801 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1861 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1921 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1981 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2041 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2101 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2161 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     2221 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2281 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2341 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     2401 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     2461 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     2521 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     2581 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2641 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2701 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2761 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2821 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2881 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2941 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     3001 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     3061 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3121 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3181 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3241 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3301 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3361 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3421 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3481 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3541 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3601 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3661 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3721 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3781 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3841 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3901 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3961 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4021 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4081 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4141 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4201 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     4261 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     4321 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     4381 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     4441 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     4501 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     4561 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     4621 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4681 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4741 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4801 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4861 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4921 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4981 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     5041 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5101 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5161 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5221 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5281 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5341 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5401 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     5461 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5521 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5581 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5641 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5701 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5761 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5821 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5881 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5941 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     6001 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6061 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6121 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6181 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6241 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6301 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6361 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatg
//
