LOCUS       sequence-218835-        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"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     141..158
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     142..158
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     165..184
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        165..183
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     216..232
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     216..232
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1224..1925
                     /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(1357..1375)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1625..1644
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1819..1842
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     promoter        complement(3184..3202)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3223..3239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3223..3239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3236..3258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3247..3263
                     /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(3271..3277,3278..3295,3296..3301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3316..3337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3454..3471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3625..4213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3705..3724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4384..5175,5176..5244))
                     /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     5007..5026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5245..5349)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5375..5803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5462..5481)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5672..5693
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      121 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
      181 agggcgaatt ggagctccac cgcggtggcg gccgctctag aactagtgga tccgggccca
      241 cactaaagtt agagaaccac aggctcgctc acaaccctga cttctccatg tcagttccga
      301 tctttgcgaa ccgcagacag ggaaggtctt ctctcagggg tcatgcccgc ggccgccctc
      361 cacggcgagg tccgcactcg cgcagccggc cccgcggccg cctcacctgg tcgcacacta
      421 ccacgtcgaa ctcctcgtcg gcgaggaaca gcacgtagag cgccaggaaa accatgcgca
      481 cgtaggcgca gacggcggcg ccgcggccgc cccagcccag gcctcgcggc agccagtccc
      541 cggcacagcg caccggtagc tcgcggctct cggcgaaaca gtggcccggg tcgtagtgcg
      601 ctgtccagat cttcacgcta cacccgcgcg cctgcagcgc cagcgccgcg tccaacacca
      661 gccgctcagc gccgcccacg cccaggtctg ggtggaggaa cagcaccgac ggcttgggaa
      721 ccgagtcccg ttcccggccc tgctcctccg ccatggccct ggagccgcaa ctgcaccccg
      781 caccctgatg ggggtcttct gcgcaagctc cgcgctcgta gctcccagct ggccactgcg
      841 ggccgacccc gccctgccgt acgtgcgtca gttaggccac atcagcgcaa atctgtgagg
      901 gtctagtaac tgcctgagaa aatatcttgt ctgaccccgg ttatattttt ccttcggtag
      961 ggattggact ttctgaagga cgttgtgatc caaaggaagg aggccggagg tctctacttc
     1021 ccatacagca ggtaactaag ttgtctgtag cagactgtct acaggcatat cgtgagacga
     1081 cccaggcgtc cctggggtca gagaggacct tgcctgcaag tccgggggcg gggcctgagt
     1141 cagtctcgcc agctgccggt ctttcggggg ctccgtaact ttctatccgt ccgcgtcagc
     1201 gccttgccac actcatctcc aatatggtga gcaagggcga ggaggtcatc aaagagttca
     1261 tgcgcttcaa ggtgcgcatg gagggctcca tgaacggcca cgagttcgag atcgagggcg
     1321 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg
     1381 gccccctgcc cttcgcctgg gacatcctgt ccccccagtt catgtacggc tccaaggcgt
     1441 acgtgaagca ccccgccgac atccccgatt acaagaagct gtccttcccc gagggcttca
     1501 agtgggagcg cgtgatgaac ttcgaggacg gcggtctggt gaccgtgacc caggactcct
     1561 ccctgcagga cggcacgctg atctacaagg tgaagatgcg cggcaccaac ttcccccccg
     1621 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc
     1681 cccgcgacgg cgtgctgaag ggcgagatcc accaggccct gaagctgaag gacggcggcc
     1741 actacctggt ggagttcaag accatctaca tggccaagaa gcccgtgcaa ctgcccggct
     1801 actactacgt ggacaccaag ctggacatca cctcccacaa cgaggactac accatcgtgg
     1861 aacagtacga gcgctccgag ggccgccacc acctgttcct gtacggcatg gacgagctgt
     1921 acaagagcgg cctgagaagc atggtatggc ggcccttcca tgatccccgc ctctcccaga
     1981 agccctgact cctcctgctt tgcgccgtgc ttttcctctg tagctccctt gcttccccca
     2041 gcctcgggtg tgggtgtcta ggccggggtt ctggggcagg cctgccgcgc tcacccgtct
     2101 gtctgcttgt ctccctctac agcctggtcc gacccccagt ggcactaacg tgggatcctc
     2161 agggcgctct cccagcaaag cagtggccgc ccgggcggcg ggatccactg tccggcagag
     2221 gtaaggaacc ctgcagttcg ttcgcttcca gactcggaga taggacccag aacctcgctg
     2281 attctggggt ggagacccta gcatgtgaag attgacaaag gcaaaatgag cttctagtga
     2341 cgtggccgtg ggagtagtta aaggcctttt gggaggaagg cgacattttt tttctcgttg
     2401 ctcagtttag ggcactactc ttaaaaaagg aaagttaaca aactggaata gagtcagaga
     2461 taactttgag aaaaccgatg tcattaaact ggtgtctctg gacctgaggt ttgcactcac
     2521 atttccatct ggcggcccca taagcaatct gtcctacaga taactcgtcc tacacaaaac
     2581 ttagtctctt ttcagctcag ctctctcact ctcaattata tctccttact tccatatggc
     2641 actgttgtac actcatttac tcagagccag aaacgtcagc gtcatcttgg atttttctta
     2701 tgctctttct ctctctagtc atatgccaga ctttaaactc tgcttgaaag ctttctcata
     2761 agctctttcc ttttcccttt ctactgcttt gcatttgcta cttaaccctt ttcttcaggc
     2821 tgtttgcttt ccagtccatc gttcgctctg ctgttactct tctgcgtagt ttctgttact
     2881 tgttgctgaa catcccaatg gcgctttgag gaagatgaac tgagatgtgt cagggtgact
     2941 tatttccatc atcgtcctta ggggagcttg ggtaggggca aggcgtgtag ctgggaccta
     3001 ggtccagacc cctggctctg ccactgaacg gctcaggtgc tttgggcagt tactccccgg
     3061 cctcactttg cacgtgtgct tacctagtgg agacaaaagt acatacctcg gtagagcgcg
     3121 cacgcctgta accccaggtt aactctctaa cctcgagggg gggcccggta ccagcttttg
     3181 ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt
     3241 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3301 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3361 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3421 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3481 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3541 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3601 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3661 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3721 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3781 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3841 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3901 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3961 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4021 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4081 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4141 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4201 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4261 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4321 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4381 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4441 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4501 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4561 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4621 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4681 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4741 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4801 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4861 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4921 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4981 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5041 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5101 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5161 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5221 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5281 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5341 ggttccgcgc acatttcccc gaaaagtgcc acctacgcgc cctgtagcgg cgcattaagc
     5401 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5461 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5521 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5581 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5641 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5701 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5761 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5821 cttacaattt
//
