LOCUS       sequence-481760-        4245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Arabinose-inducible plasmid with ALFA and Twin-Strep tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4245)
  AUTHORS   Reddy KD, Rasool B, Akher FB, Kutlesic N, Pant S, Boudker O
  TITLE     Evolutionary analysis reveals the origin of sodium coupling in 
            glutamate transporters.
  JOURNAL   Nat Struct Mol Biol (2025).
  PUBMED    38106174
REFERENCE   2  (bases 1 to 4245)
  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: "Nat Struct 
            Mol Biol (2025)."
COMMENT     Sequence Label: pBAD24-ALFA-TwinStrep
FEATURES             Location/Qualifiers
     source          1..4245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     117..134
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    149..289
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(215..234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     375..397
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(553..1431)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1458..1742
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1665..1684
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1801..1818
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1843..1881
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             join(1906..1911,1912..1935,1936..1971,1972..1995)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             2020..2049
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2103..2120)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2273..2359
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2451..2478
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2497..2588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2589..2657,2658..3449)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2807..2826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3620..4208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4109..4128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
       61 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
      121 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
      181 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
      241 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
      301 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
      361 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
      421 cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg catgcataat gtgcctgtca
      481 aatggacgaa gcagggattc tgcaaaccct atgctactcc gtcaagccgt caattgtctg
      541 attcgttacc aattatgaca acttgacggc tacatcattc actttttctt cacaaccggc
      601 acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg
      661 atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag
      721 cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg
      781 ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc
      841 gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg
      901 attatccatc ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg
      961 ctcaagcaga tttatcgcca gcagctccga atagcgccct tccccttgcc cggcgttaat
     1021 gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc
     1081 cgtattggca aatattgacg gccagttaag ccattcatgc cagtaggcgc gcggacgaaa
     1141 gtaaacccac tggtgatacc attcgcgagc ctccggatga cgaccgtagt gatgaatctc
     1201 tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc cctgattttt
     1261 caccaccccc tgaccgcgaa tggtgagatt gagaatataa cctttcattc ccagcggtcg
     1321 gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc gttaaacccg ccaccagatg
     1381 ggcattaaac gagtatcccg gcagcagggg atcattttgc gcttcagcca tacttttcat
     1441 actcccgcca ttcagagaag aaaccaattg tccatattgc atcagacatt gccgtcactg
     1501 cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta aaagcattct
     1561 gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg
     1621 cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca tagcattttt
     1681 atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta ctgtttctcc
     1741 atacccgttt ttttgggcta gcaggaggaa ttcaccatgg tacccccggg ctcgagctcc
     1801 ctggtgccgc gtggtagcgg tggcggtagc ggcggtagcc cgagccgtct ggaggaagag
     1861 ctgcgtcgtc gtctgaccga accgggcggt ggcagcggtg gcggtagcgc gtggagccac
     1921 ccgcagtttg agaagggcgg tggcagcggt ggcggtagcg gcggtagcgc ttggtctcac
     1981 ccgcaatttg aaaaaggcgg tggcagcggt ggcggtagcc atcatcacca tcaccaccat
     2041 caccatcatt gataatctag atctaagctt ggctgttttg gcggatgaga gaagattttc
     2101 agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc
     2161 ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc
     2221 gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa
     2281 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgc cggtgaacgc
     2341 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg
     2401 agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat
     2461 cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt
     2521 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2581 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2641 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2701 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2761 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2821 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2881 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2941 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3001 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3061 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3121 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3181 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3241 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3301 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3361 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3421 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3481 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3541 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3601 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3661 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3721 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3781 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3841 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3901 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3961 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4021 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4081 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4141 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4201 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggcct
//
