LOCUS       sequence-211948-        6382 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of biotin ligase (BirA) with an N-terminal myc 
            epitope.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6382)
  AUTHORS   Buser DP, Schleicher KD, Prescianotto-Baschong C, Spiess M
  TITLE     A versatile nanobody-based toolkit to analyze retrograde transport 
            from the cell surface.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Jul 3;115(27):E6227-E6236. doi: 
            10.1073/pnas.1801865115. Epub 2018 Jun 18.
  PUBMED    29915061
REFERENCE   2  (bases 1 to 6382)
  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: 
            "10.1073/pnas.1801865115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-07-3- 3"; volume: "115"; issue: "27"; pages: 
            "E6227-E6236"
COMMENT     Sequence Label: pET21d-myc-BirA
FEATURES             Location/Qualifiers
     source          1..6382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(48..1130)
                     /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     1092..1111
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1131..1208)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1414..1433
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1517..1536
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1517..1535
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1536..1560
                     /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             1575..1597
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1613..1642
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1643..2608
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1643..2605
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2630..2647
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2700..2718)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2714..2761
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2798..3253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2885..2904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3095..3116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3279..3383
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3384..3452,3453..4244)
                     /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(3602..3621)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4415..5003
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4904..4923
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5157..5174
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5189..5328
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5254..5273)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5414..5436
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5430..5621)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
       61 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
      121 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
      181 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
      241 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
      301 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
      361 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
      421 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
      481 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
      541 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
      601 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
      661 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
      721 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
      781 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
      841 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
      901 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
      961 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     1021 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     1081 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     1141 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     1201 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     1261 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     1321 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     1381 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     1441 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     1501 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     1561 cctctagaaa taattttgtt taactttaag aaggagatat accatggcta gcgaacagaa
     1621 gctcatctcc gaagaggatc tcatgaagga taacaccgtg ccactgaaat tgattgccct
     1681 gttagcgaac ggtgaatttc actctggcga gcagttgggt gaaacgctgg gaatgagccg
     1741 ggcggctatt aataaacaca ttcagacact gcgtgactgg ggcgttgatg tctttaccgt
     1801 tccgggtaaa ggatacagcc tgcctgagcc tatccagtta cttaatgcta aacagatatt
     1861 gggtcagctg gatggcggta gtgtagccgt gctgcctgtg attgactcca cgaatcagta
     1921 ccttcttgat cgtatcggag agcttaaatc gggcgatgct tgcattgcag aataccagca
     1981 ggctggccgt ggtcgccggg gtcggaaatg gttttcgcct tttggcgcaa acttatattt
     2041 gtcgatgttc tggcgtctgg aacaaggccc ggcggcggcg attggtttaa gtctggttat
     2101 cggtatcgtg atggcggaag tattacgcaa gctgggtgca gataaagttc gtgttaaatg
     2161 gcctaatgac ctctatctgc aggatcgcaa gctggcaggc attctggtgg agctgactgg
     2221 caaaactggc gatgcggcgc aaatagtcat tggagccggg atcaacatgg caatgcgccg
     2281 tgttgaagag agtgtcgtta atcaggggtg gatcacgctg caggaagcgg ggatcaatct
     2341 cgatcgtaat acgttggcgg ccatgctaat acgtgaatta cgtgctgcgt tggaactctt
     2401 cgaacaagaa ggattggcac cttatctgtc gcgctgggaa aagctggata attttattaa
     2461 tcgcccagtg aaacttatca ttggtgataa agaaatattt ggcatttcac gcggaataga
     2521 caaacagggg gctttattac ttgagcagga tggaataata aaaccctgga tgggcggtga
     2581 aatatccctg cgtagtgcag aaaaatgaaa gcttgcggcc gcactcgagc accaccacca
     2641 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     2701 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     2761 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     2821 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2881 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2941 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3001 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3061 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3121 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3181 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3241 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3301 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3361 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3421 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3481 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3541 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3601 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3661 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3721 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3781 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3841 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3901 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3961 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4021 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4081 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4141 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4201 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4261 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4321 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4381 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4441 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4501 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4561 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4621 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4681 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4741 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4801 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4861 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4921 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4981 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5041 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5101 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5161 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5221 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5281 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     5341 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5401 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5461 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     5521 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5581 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5641 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5701 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5761 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5821 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5881 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5941 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     6001 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     6061 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     6121 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     6181 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     6241 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     6301 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     6361 gctctcaagg gcatcggtcg ag
//
