LOCUS       sequence-218834-        5830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert dTomato at the SEC61B locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5830)
  AUTHORS   Iyer S, Mir A, Vega-Badillo J, Roscoe BP, Ibraheim R, Zhu LJ, Lee J,
            Liu P, Luk K, Mintzer E, Guo D, Soares de Brito J, Emerson CP Jr, 
            Zamore PD, Sontheimer EJ, Wolfe SA
  TITLE     Efficient Homology-Directed Repair with Circular Single-Stranded DNA
            Donors.
  JOURNAL   CRISPR J. 2022 Oct;5(5):685-701. doi: 10.1089/crispr.2022.0058. Epub
            2022 Sep 7.
  PUBMED    36070530
REFERENCE   2  (bases 1 to 5830)
  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: 
            "10.1089/crispr.2022.0058"; journalName: "CRISPR J"; date: 
            "2022-10"; volume: "5"; issue: "5"; pages: "685-701"
COMMENT     Sequence Label: pBS-f1(+)-SEC61B-dTomato
FEATURES             Location/Qualifiers
     source          1..5830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1283..1301)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1322..1338)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1322..1338)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1335..1357)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1346..1362
                     /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)."
     promoter        complement(join(1370..1376,1377..1394,1395..1400))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1415..1436
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1553..1570)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1724..2312)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1804..1823)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2483..3274,3275..3343))
                     /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     3106..3125
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3344..3448)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3474..3929)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3611..3632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3823..3842
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4056..4078
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4070..4087
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4071..4087
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4094..4113
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4094..4112
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4145..4161
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4145..4161
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(5153..5830,1..24)
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(5286..5304)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5554..5573
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5748..5771
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
ORIGIN
        1 tacggcatgg acgagctgta caagagcggc ctgagaagca tggtatggcg gcccttccat
       61 gatccccgcc tctcccagaa gccctgactc ctcctgcttt gcgccgtgct tttcctctgt
      121 agctcccttg cttcccccag cctcgggtgt gggtgtctag gccggggttc tggggcaggc
      181 ctgccgcgct cacccgtctg tctgcttgtc tccctctaca gcctggtccg acccccagtg
      241 gcactaacgt gggatcctca gggcgctctc ccagcaaagc agtggccgcc cgggcggcgg
      301 gatccactgt ccggcagagg taaggaaccc tgcagttcgt tcgcttccag actcggagat
      361 aggacccaga acctcgctga ttctggggtg gagaccctag catgtgaaga ttgacaaagg
      421 caaaatgagc ttctagtgac gtggccgtgg gagtagttaa aggccttttg ggaggaaggc
      481 gacatttttt ttctcgttgc tcagtttagg gcactactct taaaaaagga aagttaacaa
      541 actggaatag agtcagagat aactttgaga aaaccgatgt cattaaactg gtgtctctgg
      601 acctgaggtt tgcactcaca tttccatctg gcggccccat aagcaatctg tcctacagat
      661 aactcgtcct acacaaaact tagtctcttt tcagctcagc tctctcactc tcaattatat
      721 ctccttactt ccatatggca ctgttgtaca ctcatttact cagagccaga aacgtcagcg
      781 tcatcttgga tttttcttat gctctttctc tctctagtca tatgccagac tttaaactct
      841 gcttgaaagc tttctcataa gctctttcct tttccctttc tactgctttg catttgctac
      901 ttaacccttt tcttcaggct gtttgctttc cagtccatcg ttcgctctgc tgttactctt
      961 ctgcgtagtt tctgttactt gttgctgaac atcccaatgg cgctttgagg aagatgaact
     1021 gagatgtgtc agggtgactt atttccatca tcgtccttag gggagcttgg gtaggggcaa
     1081 ggcgtgtagc tgggacctag gtccagaccc ctggctctgc cactgaacgg ctcaggtgct
     1141 ttgggcagtt actccccggc ctcactttgc acgtgtgctt acctagtgga gacaaaagta
     1201 catacctcgg tagagcgcgc acgcctgtaa ccccaggtta actctctaac ctcgaggggg
     1261 ggcccggtac cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat
     1321 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     1381 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1441 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1501 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1561 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1621 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1681 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1741 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1801 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1861 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1921 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1981 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2041 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2101 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2161 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2221 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2281 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2341 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2401 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2461 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2521 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2581 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     2641 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2701 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2761 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     2821 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     2881 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     2941 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3001 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3061 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3121 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3181 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3241 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3301 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3361 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3421 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     3481 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3541 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3601 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3661 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3721 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3781 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3841 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3901 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     3961 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     4021 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     4081 ggccagtgaa ttgtaatacg actcactata gggcgaattg gagctccacc gcggtggcgg
     4141 ccgctctaga actagtggat ccgggcccac actaaagtta gagaaccaca ggctcgctca
     4201 caaccctgac ttctccatgt cagttccgat ctttgcgaac cgcagacagg gaaggtcttc
     4261 tctcaggggt catgcccgcg gccgccctcc acggcgaggt ccgcactcgc gcagccggcc
     4321 ccgcggccgc ctcacctggt cgcacactac cacgtcgaac tcctcgtcgg cgaggaacag
     4381 cacgtagagc gccaggaaaa ccatgcgcac gtaggcgcag acggcggcgc cgcggccgcc
     4441 ccagcccagg cctcgcggca gccagtcccc ggcacagcgc accggtagct cgcggctctc
     4501 ggcgaaacag tggcccgggt cgtagtgcgc tgtccagatc ttcacgctac acccgcgcgc
     4561 ctgcagcgcc agcgccgcgt ccaacaccag ccgctcagcg ccgcccacgc ccaggtctgg
     4621 gtggaggaac agcaccgacg gcttgggaac cgagtcccgt tcccggccct gctcctccgc
     4681 catggccctg gagccgcaac tgcaccccgc accctgatgg gggtcttctg cgcaagctcc
     4741 gcgctcgtag ctcccagctg gccactgcgg gccgaccccg ccctgccgta cgtgcgtcag
     4801 ttaggccaca tcagcgcaaa tctgtgaggg tctagtaact gcctgagaaa atatcttgtc
     4861 tgaccccggt tatatttttc cttcggtagg gattggactt tctgaaggac gttgtgatcc
     4921 aaaggaagga ggccggaggt ctctacttcc catacagcag gtaactaagt tgtctgtagc
     4981 agactgtcta caggcatatc gtgagacgac ccaggcgtcc ctggggtcag agaggacctt
     5041 gcctgcaagt ccgggggcgg ggcctgagtc agtctcgcca gctgccggtc tttcgggggc
     5101 tccgtaactt tctatccgtc cgcgtcagcg ccttgccaca ctcatctcca atatggtgag
     5161 caagggcgag gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg agggctccat
     5221 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     5281 gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc
     5341 cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca tccccgatta
     5401 caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     5461 cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga tctacaaggt
     5521 gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga agaccatggg
     5581 ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca
     5641 ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga ccatctacat
     5701 ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc tggacatcac
     5761 ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg gccgccacca
     5821 cctgttcctg
//
