LOCUS       sequence-383965-        5546 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector encoding 6xHis-AviTag-ALFAtag-eGFP. 
            Co-expression in cells with biotin ligase (BirA) results in 
            efficient biotinylation of the AviTag sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5546)
  AUTHORS   Sloas DC, Tran JC, Marzilli AM, Ngo JT
  TITLE     Tension-tuned receptors for synthetic mechanotransduction and 
            intercellular force detection.
  JOURNAL   Nat Biotechnol. 2023 Jan 16. doi: 10.1038/s41587-022-01638-y.
  PUBMED    36646932
REFERENCE   2  (bases 1 to 5546)
  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 
            Biotechnol. 2023 Jan 16. doi: 10.1038/s41587-022-01638-y."
COMMENT     Sequence Label: pBAD-6xHis-AviTag-ALFAtag-eGFP
FEATURES             Location/Qualifiers
     source          1..5546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..47
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    62..202
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(128..147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     288..310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     448..467
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(647..1525)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1552..1836
                     /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     1759..1778
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1863..1885
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1905..1922
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1935..1979
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1992..2030
                     /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             2046..2075
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="ASMTGGQQMG"
     CDS             2088..2108
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2115..2117,2118..2120,2121..2834)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2115..2117,2118..2120,2121..2831)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2160..2181)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2421..2440)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2768..2789
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2880..2897)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3050..3136
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3228..3255
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3274..3365
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3366..3434,3435..4226)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3584..3603)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4268..4723
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4355..4374)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4565..4586
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      4834..5422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5323..5342
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
       61 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atctggtgca
      121 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
      181 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
      241 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
      301 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagaaggaga tggcgcccaa
      361 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
      421 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
      481 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatct gctcatgttt
      541 gacagcttat catcgatgca taatgtgcct gtcaaatgga cgaagcaggg attctgcaaa
      601 ccctatgcta ctccgtcaag ccgtcaattg tctgattcgt taccaattat gacaacttga
      661 cggctacatc attcactttt tcttcacaac cggcacggaa ctcgctcggg ctggccccgg
      721 tgcatttttt aaatacccgc gagaaataga gttgatcgtc aaaaccaaca ttgcgaccga
      781 cggtggcgat aggcatccgg gtggtgctca aaagcagctt cgcctggctg atacgttggt
      841 cctcgcgcca gcttaagacg ctaatcccta actgctggcg gaaaagatgt gacagacgcg
      901 acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg tctgccaggt
      961 gatcgctgat gtactgacaa gcctcgcgta cccgattatc catcggtgga tggagcgact
     1021 cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag cagatttatc gccagcagct
     1081 ccgaatagcg cccttcccct tgcccggcgt taatgatttg cccaaacagg tcgctgaaat
     1141 gcggctggtg cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt gacggccagt
     1201 taagccattc atgccagtag gcgcgcggac gaaagtaaac ccactggtga taccattcgc
     1261 gagcctccgg atgacgaccg tagtgatgaa tctctcctgg cgggaacagc aaaatatcac
     1321 ccggtcggca aacaaattct cgtccctgat ttttcaccac cccctgaccg cgaatggtga
     1381 gattgagaat ataacctttc attcccagcg gtcggtcgat aaaaaaatcg agataaccgt
     1441 tggcctcaat cggcgttaaa cccgccacca gatgggcatt aaacgagtat cccggcagca
     1501 ggggatcatt ttgcgcttca gccatacttt tcatactccc gccattcaga gaagaaacca
     1561 attgtccata ttgcatcaga cattgccgtc actgcgtctt ttactggctc ttctcgctaa
     1621 ccaaaccggt aaccccgctt attaaaagca ttctgtaaca aagcgggacc aaagccatga
     1681 caaaaacgcg taacaaaagt gtctataatc acggcagaaa agtccacatt gattatttgc
     1741 acggcgtcac actttgctat gccatagcat ttttatccat aagattagcg gatactacct
     1801 gacgcttttt atcgcaactc tctactgttt ctccataccc gtttttttgg gctagaaata
     1861 attttgttta actttaagaa ggagatatac atatgcgggg ttctcatcat catcatcatc
     1921 atggtatggc tagcggtctg aacgacatct ttgaagcgca gaaaatcgaa tggcacgaag
     1981 gctctggtgg ttctcgtctg gaagaagaac tgcgtcgtcg tctgaccgaa ggttctggca
     2041 ccaaagctag catgactggt ggacagcaaa tgggtcggga tctgtacgag aacctgtact
     2101 tccagggctc gagcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     2161 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2221 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     2281 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     2341 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2401 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2461 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2521 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2581 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2641 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2701 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2761 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2821 agctgtacaa gtaagaattc gaagcttggc tgttttggcg gatgagagaa gattttcagc
     2881 ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc
     2941 agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc
     3001 gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg
     3061 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     3121 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg
     3181 gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct
     3241 gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa
     3301 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3361 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3421 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3481 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3541 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3601 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3661 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3721 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3781 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3841 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3901 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3961 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4021 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4081 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4141 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4201 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4261 ttgatttacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4321 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4381 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4441 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4501 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4561 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     4621 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4681 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttaaaagga tctaggtgaa
     4741 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4801 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4861 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4921 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4981 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5041 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5101 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5161 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5221 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5281 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5341 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5401 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5461 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5521 tattaccgcc tttgagtgag ctgata
//
