LOCUS       sequence-375723-        5945 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Arf1 (aa17-181, Q71L mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5945)
  AUTHORS   Ren X, Farias GG, Canagarajah BJ, Bonifacino JS, Hurley JH
  TITLE     Structural basis for recruitment and activation of the AP-1 clathrin
            adaptor complex by Arf1.
  JOURNAL   Cell. 2013 Feb 14;152(4):755-67. doi: 10.1016/j.cell.2012.12.042.
  PUBMED    23415225
REFERENCE   2  (bases 1 to 5945)
  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"; doi: 
            "10.1016/j.cell.2012.12"; journalName: "Cell"; date: "2013-02-14- 
            14"; volume: "152"; issue: "4"; pages: "755-67"
COMMENT     Sequence Label: pHis2-Arf1 (17-181)-Q71L
FEATURES             Location/Qualifiers
     source          1..5945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             118..135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(188..206)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      202..249
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      286..741
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(373..392)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     583..604
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        767..871
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(872..940,941..1732)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1090..1109)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1903..2491
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2392..2411
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2645..2662
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2677..2816
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2742..2761)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2902..2924
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2918..3109)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3918..5000)
                     /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     4962..4981
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5001..5078)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5284..5303
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5387..5406
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5387..5405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5406..5430
                     /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             5445..5467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5487..5504
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5526..5546
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 cactcactac gccacaggaa ctggtacatt caggccacct gcgccaccag cggcgacggg
       61 ctctatgaag gactggactg gctgtccaat cagctccgga accagaagtg actcgagcac
      121 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      181 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      241 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      301 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      361 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      421 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      481 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
      541 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
      601 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
      661 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
      721 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
      781 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
      841 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
      901 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      961 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1021 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1081 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     1141 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1201 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1261 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1321 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1381 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     1441 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1501 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1561 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1621 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1681 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1741 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1801 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1861 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1921 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1981 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2041 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2101 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2161 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2221 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2281 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2341 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2401 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2461 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2521 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2581 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     2641 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     2701 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     2761 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     2821 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2881 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2941 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3001 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3061 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3121 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3181 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     3241 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     3301 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     3361 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     3421 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     3481 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     3541 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     3601 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     3661 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     3721 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     3781 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     3841 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     3901 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3961 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4021 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4081 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4141 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     4201 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4261 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     4321 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     4381 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     4441 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     4501 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     4561 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     4621 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     4681 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     4741 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     4801 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     4861 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     4921 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     4981 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5041 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     5101 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     5161 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     5221 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     5281 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     5341 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     5401 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     5461 aaggagatat acatatgtcg tactaccatc accatcacca tcacgattac gatatcccaa
     5521 cgaccgaaaa cctgtatttt cagggcgcca tgggatccga aatgcgcatc ctcatggtgg
     5581 gcctggatgc tgcagggaag accacgatcc tctacaagct taagctgggt gagatcgtga
     5641 ccaccattcc caccataggc ttcaacgtgg aaaccgtgga gtacaagaac atcagcttca
     5701 ctgtgtggga cgtgggtggc ctggacaaga tccggcccct gtggcgccac tacttccaga
     5761 acacacaagg cctgatcttc gtggtggaca gcaatgacag agagcgtgtg aacgaggccc
     5821 gtgaggagct catgaggatg ctggccgagg acgagctccg ggatgctgtc ctcctggtgt
     5881 tcgccaacaa gcaggacctc cccaacgcca tgaatgcggc cgagatcaca gacaagctgg
     5941 ggctg
//
