LOCUS       sequence-368309-        6238 bp    DNA     circular SYN 14-APR-2025
DEFINITION  CAG promoter-driven expression of TurboID-fused protein (N 
            terminus).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6238)
  AUTHORS   Oura S, Ninomiya A, Sugihara F, Matzuk MM, Ikawa M
  TITLE     Proximity-dependent biotin labeling in testicular germ cells 
            identified TESMIN-associated proteins.
  JOURNAL   Sci Rep. 2022 Dec 23;12(1):22198. doi: 10.1038/s41598-022-26501-7.
  PUBMED    36564444
REFERENCE   2  (bases 1 to 6238)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2022-12-23"; volume: "12(1):22198. doi"; pages: " 
            10.1038/s41598-022-26501-7"
COMMENT     Sequence Label: pCAG-3xFLAG-TurboID-MCS
FEATURES             Location/Qualifiers
     source          1..6238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        17..396
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          675..1689
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1615..1637
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1697..1716
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1753..1818
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1834..2790
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     primer_bind     complement(2918..2937)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2983..3038
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3037..3056)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3393..3412)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3394..3412)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3422..3439)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3422..3438)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3431..3453)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3580..4035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3667..3686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3877..3898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4061..4165
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4166..4234,4235..5026)
                     /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"
     rep_origin      5197..5785
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5686..5705
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5939..5956
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6073..6094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6109..6114,6115..6132,6133..6139)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6147..6163
                     /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     6152..6174
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6171..6187
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6171..6187
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6206..6226
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6208..6226
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gagctcatcg atactcgaca ttgattattg actagttatt aatagtaatc aattacgggg
       61 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      121 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      181 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      241 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      301 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      361 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      421 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      481 ttattttgtg cagcgatggg ggcggggggg gggggggnsg cgcgccrggs ggggsggggs
      541 ggggsgrggg gsggggsggg gsgrggcggr gaggtgcggc ggcagccaat cagagcggcg
      601 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
      661 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
      721 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
      781 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc
      841 tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct
      901 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg
      961 gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg
     1021 ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct
     1081 gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc
     1141 aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc
     1201 tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg
     1261 tgccgggcgg ggcggggccg cctcgggccg gggrgggctc gggggagggg cgcggcggcc
     1321 cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat
     1381 cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga
     1441 ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg
     1501 aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc
     1561 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     1621 gcttctggcg tgtgaccggc ggctctagcc tctgctaacc atgttcatgc cttcttcttt
     1681 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaatct
     1741 agagccgcca tggactacaa agaccatgac ggtgattata aagatcatga cattgattac
     1801 aaggatgacg atgacaaggg gggcggggct agcaaagaca atactgtgcc tctgaagctg
     1861 atcgctctcc tggctaatgg cgagttccat agtggcgaac agctgggaga aaccctgggc
     1921 atgtccaggg ccgctatcaa caagcacatt cagactctgc gcgactgggg cgtggacgtg
     1981 ttcaccgtgc ccggaaaggg ctactctctg cccgagccta tcccgctgct gaacgctaaa
     2041 cagattctgg gacagctgga cggcgggagc gtggcagtcc tgcctgtggt cgactccacc
     2101 aatcagtacc tgctggatcg aatcggcgag ctgaagagtg gggatgcttg cattgcagaa
     2161 tatcagcagg cagggagagg aagcagaggg aggaaatggt tctctccttt tggagctaac
     2221 ctgtacctga gtatgttttg gcgcctgaag cggggaccag cagcaatcgg cctgggcccg
     2281 gtcatcggaa ttgtcatggc agaagcgctg cgaaagctgg gagcagacaa ggtgcgagtc
     2341 aaatggccca atgacctgta tctgcaggat agaaagctgg caggcatcct ggtggagctg
     2401 gccggaataa caggcgatgc tgcacagatc gtcattggcg ccgggattaa cgtggctatg
     2461 aggcgcgtgg aggaaagcgt ggtcaatcag ggctggatca cactgcagga agcagggatt
     2521 aacctggaca ggaatactct ggccgctacg ctgatccgag agctgcgggc agccctggaa
     2581 ctgttcgagc aggaaggcct ggctccatat ctgccacggt gggagaagct ggataacttc
     2641 atcaatagac ccgtgaagct gatcattggg gacaaagaga ttttcgggat tagccggggg
     2701 attgataaac agggagccct gctgctggaa caggacggag ttatcaaacc ctggatgggc
     2761 ggagaaatca gtctgcggtc tgccgaaaag ggatccctgc aggaattcga tatcaagctt
     2821 gtcgacgcgg ccgcctcgag actcctcagg tgcaggctgc ctatcagaag gtggtggctg
     2881 gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt
     2941 atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt
     3001 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     3061 caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata
     3121 tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc
     3181 ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat
     3241 tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag
     3301 ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgaa
     3361 gatccctcga cttaattaag gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     3421 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     3481 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     3541 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     3601 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3661 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3721 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3781 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3841 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3901 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3961 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4021 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4081 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4141 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     4201 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4261 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     4321 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     4381 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     4441 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     4501 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     4561 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     4621 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     4681 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     4741 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     4801 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     4861 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     4921 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4981 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     5041 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5101 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5161 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5221 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5281 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5341 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5401 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5461 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5521 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5581 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5641 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5701 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5761 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5821 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5881 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5941 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     6001 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     6061 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     6121 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     6181 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctg
//
