LOCUS       sequence-190141-        6495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for generation of BirA tagged strains using CRISPR tagging 
            technology.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6495)
  TITLE     pLinker-AID-Ty-HXGPRT-LoxP
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6495)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pLinker-BirA-3xHA-HXGPRT-loxP
FEATURES             Location/Qualifiers
     source          1..6495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(7..40)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1977..2010)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2054..2073)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2055..2073)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2080..2097)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2080..2096)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2089..2111)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2238..2693
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2325..2344)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2535..2556
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2719..2823
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2824..2892,2893..3684)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3042..3061)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3855..4443
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4344..4363
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4597..4614
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4731..4752
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4767..4772,4773..4790,4791..4797)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4805..4821
                     /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)."
     primer_bind     4810..4832
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4829..4845
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4829..4845
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4866..4884
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             join(4992..5339,5340..5342,5343..5951)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             5958..5984
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5988..6014
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6015..6041
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     6476..6492
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6477..6493
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aagcttataa cttcgtatag catacattat acgaagttat cagcacgaaa ccttgcattc
       61 aaacccgccc gcggaagatc cgatcttgct gctgttcgca gtcccagtag cgtcctgtcg
      121 gccgcgccgt ctctgttggt gggcagccgc tacacctgtt atctgactgc cgtgcgcgaa
      181 aatgacgcca tttttgggaa aatcggggaa cttcattctt taaaagtatg cggaggtttc
      241 ctttttcttc tgttcgtttc tttttctcgg gtttgataac cgtgttcgat gtaagcactt
      301 tccgtctctc ctccgtgctt tgttcgacat cgagaccagg tgtgcagatc cttcgcttgt
      361 cgatccggag acgcgtgtct cgtagaacct tttcatttta ccacacggca gtgcggagca
      421 ctgctctgag tgcagcaggg acgggtgaag tttcgcttta gtagtgcgtt tctgctctac
      481 ggggcgttgt cagatccagc aaaatggcgt ccaaacccat tgaagactac ggcaagggca
      541 agggccgtat tgagcccatg tatatccccg acaacacctt ctacaacgct gatgactttc
      601 ttgtgccccc ccactgcaag ccctacattg acaaaatcct cctccctggt ggattggtca
      661 aggacagagt tgagaagttg gcgtatgaca tccacagaac ttacttcggc gaggagttgc
      721 acatcatttg catcctgaaa ggctctcgcg gcttcttcaa ccttctgatc gactaccttg
      781 ccaccataca gaagtacagt ggtcgtgagt ccagcgtgcc ccccttcttc gagcactatg
      841 tccgcctgaa gtcctaccag aacgacaaca gcacaggcca gctcaccgtc ttgagcgacg
      901 acttgtcaat ctttcgcgac aagcacgttc tgattgttga ggacatcgtc gacaccggtt
      961 tcaccctcac cgagttcggt gagcgcctga aagccgtcgg tcccaagtcg atgagaatcg
     1021 ccaccctcgt cgagaagcgc acagatcgct ccaacagctt gaagggcgac ttcgtcggct
     1081 tcagcattga agacgtctgg atcgttggtt gctgctacga cttcaacgag atgttccgcg
     1141 acttcgacca cgtcgccgtc ctgagcgacg ccgctcgcaa aaagttcgag aagtaaaccc
     1201 tgcatagccc acagaagctg cccgtctctc gttttcctct cttttcggag ggatcaggga
     1261 gagtgcctcg gatcggagag agctgacgag ggggtgccag agacccctgt gtcctttatc
     1321 gaagaaaagg gatgactctt catgtggcat ttcacacagt ctcacctcgc cttgttttct
     1381 ttttgtcaat cagaacgaaa gcgagttgcg ggtgacgcag atgtgcgtgt atccactcgt
     1441 gaatgcgtta tcgttctgta tgccgctaga gtgctggact gttgctgtct gcccacgaca
     1501 gcagacaact ttccttctat gcacttgcag gatggtgcag cgcaaacgac ggagagaaag
     1561 gagcaccctc tcagtttccc tacgatgtgc tgtcagtttc gactcttcac cgcgaacgat
     1621 tggcgatacg tctctgttga cttgttaggc tccgaccacg aagctccctt aactagataa
     1681 gccgcgacac ctaagtgtac accatttgca gatcgataat ctgcgaccgc tgaatccgtc
     1741 cagatcagta aaaccgcacc acctaagtgt aaaccttgtt taggtcgata aaatgctacc
     1801 aacccccacc cacaatcgag ccttgagcgt ttctgcgcac gcgttggcct acgtgacttg
     1861 ctgatgcctg cctctggcca ttcatgccag tcagtgcgca taaaaatgtg gacacagtcg
     1921 gttgacaagt gttctggcag gctacagtga caccgcggtg gagggggatc attgaaataa
     1981 cttcgtatag catacattat acgaagttat ctgcagtcta gagcggccgc caccgcggtg
     2041 gagctccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg
     2101 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2161 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2221 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2281 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2341 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2401 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2461 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2521 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2581 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2641 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2701 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2761 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2821 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2881 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2941 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3001 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3061 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3121 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3181 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3241 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3301 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3361 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3421 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3481 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3541 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3601 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3661 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3721 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3781 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3841 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3901 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3961 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4021 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4081 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4141 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4201 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4261 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4321 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4381 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4441 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4501 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4561 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4621 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     4681 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     4741 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     4801 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     4861 ctcgaaatta accctcacta aagggaacaa aagctggtac cctgtacttc caatccaatt
     4921 taattaaaat tggaagtgga ggacgggaat tcgggcccgc tagcaagggc tcgggctcga
     4981 cccagctgga caaggacaac accgtgcccc tgaagctgat cgccctgctg gccaacggcg
     5041 agttccactc tggcgagcag ctgggagaga ccctgggaat gagcagagcc gccatcaaca
     5101 agcacatcca gacactgaga gactggggag tggacgtgtt caccgtgcct ggcaagggct
     5161 acagcctgcc tgagcctatc cagctgctga acgccaagca gatcctggga cagctggatg
     5221 gcggaagcgt ggccgtgctg cctgtgatcg actccaccaa tcagtacctg ctggacagaa
     5281 tcggagagct gaagtccggc gacgcctgca tcgccgagta ccagcaggct ggcagaggag
     5341 gcagaggacg gaagtggttc agcccattcg gagccaacct gtacctgtcc atgttctgga
     5401 gactggagca gggacctgct gctgccatcg gactgagtct ggtgatcgga atcgtgatgg
     5461 ccgaggtgct gagaaagctg ggagccgaca aggtgagagt gaagtggcct aatgacctgt
     5521 acctccagga ccgcaagctg gctggcatcc tggtggagct gacaggcaag acaggcgatg
     5581 ccgctcagat cgtgatcgga gccggaatca acatggccat gagaagagtg gaggagagcg
     5641 tggtgaacca gggctggatc accctgcagg aggctggcat caacctggac cggaacaccc
     5701 tggccgccat gctgatcaga gagctgagag ccgctctgga gctgttcgag caggagggac
     5761 tggctcctta cctgagcaga tgggagaagc tggacaactt catcaacaga cctgtgaagc
     5821 tgatcatcgg cgacaaggaa atcttcggca tctccagagg aatcgacaag cagggagctc
     5881 tgctgctgga gcaggacgga atcatcaagc cctggatggg cggagaaatc tccctgagaa
     5941 gcgcagagaa gcctaggtac ccgtacgacg tcccggacta cgctggctat ccctatgatg
     6001 tgcccgatta tgcgtatcct tacgatgttc cagattatgc ctaacccggg catatgtaga
     6061 aaagttgtaa cgttagtaaa cgtaacacac gggtataata gccatttaat ggcgccgtat
     6121 atttttcact ccccctctcc gataggagga agaaagacga tccgtctgcg aatgtatatt
     6181 acatcgaccg gccatagtac gagaagtggt gccggtattg ttctgtccgg ggggtgtacc
     6241 atcctatttt ttgtatatgt cgttcttgtc ggggagcaac agcccatctt ccggctattg
     6301 tctcgtatca gttgtgaaac agactgcgtt ttgccgaaaa tgagtcctat gtccgatggc
     6361 tatttgtacc gcttcgtacc atttcgcttg tcagctgtaa tcaaccgaat tcatttggcg
     6421 agacatgatt ccgattaaat tctttactgc gcgtgctcgg atccactagt tctactcgag
     6481 gtcgacggta tcgat
//
