LOCUS       sequence-223541-        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     30..52
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     44..61
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     45..61
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     68..87
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        68..86
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     119..135
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     119..135
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1127..1828
                     /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(1260..1278)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1528..1547
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1722..1745
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     promoter        complement(3087..3105)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3126..3142)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3126..3142)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3139..3161)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3150..3166
                     /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(3174..3180,3181..3198,3199..3204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3219..3240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3357..3374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3528..4116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3608..3627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4287..5078,5079..5147))
                     /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     4910..4929
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5148..5252)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5278..5733
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5365..5384)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5575..5596
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
       61 tgaattgtaa tacgactcac tatagggcga attggagctc caccgcggtg gcggccgctc
      121 tagaactagt ggatccgggc ccacactaaa gttagagaac cacaggctcg ctcacaaccc
      181 tgacttctcc atgtcagttc cgatctttgc gaaccgcaga cagggaaggt cttctctcag
      241 gggtcatgcc cgcggccgcc ctccacggcg aggtccgcac tcgcgcagcc ggccccgcgg
      301 ccgcctcacc tggtcgcaca ctaccacgtc gaactcctcg tcggcgagga acagcacgta
      361 gagcgccagg aaaaccatgc gcacgtaggc gcagacggcg gcgccgcggc cgccccagcc
      421 caggcctcgc ggcagccagt ccccggcaca gcgcaccggt agctcgcggc tctcggcgaa
      481 acagtggccc gggtcgtagt gcgctgtcca gatcttcacg ctacacccgc gcgcctgcag
      541 cgccagcgcc gcgtccaaca ccagccgctc agcgccgccc acgcccaggt ctgggtggag
      601 gaacagcacc gacggcttgg gaaccgagtc ccgttcccgg ccctgctcct ccgccatggc
      661 cctggagccg caactgcacc ccgcaccctg atgggggtct tctgcgcaag ctccgcgctc
      721 gtagctccca gctggccact gcgggccgac cccgccctgc cgtacgtgcg tcagttaggc
      781 cacatcagcg caaatctgtg agggtctagt aactgcctga gaaaatatct tgtctgaccc
      841 cggttatatt tttccttcgg tagggattgg actttctgaa ggacgttgtg atccaaagga
      901 aggaggccgg aggtctctac ttcccataca gcaggtaact aagttgtctg tagcagactg
      961 tctacaggca tatcgtgaga cgacccaggc gtccctgggg tcagagagga ccttgcctgc
     1021 aagtccgggg gcggggcctg agtcagtctc gccagctgcc ggtctttcgg gggctccgta
     1081 actttctatc cgtccgcgtc agcgccttgc cacactcatc tccaatatgg tgagcaaggg
     1141 cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct ccatgaacgg
     1201 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     1261 caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca
     1321 gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg attacaagaa
     1381 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggtct
     1441 ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca aggtgaagat
     1501 gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca tgggctggga
     1561 ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga tccaccaggc
     1621 cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct acatggccaa
     1681 gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca tcacctccca
     1741 caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc accacctgtt
     1801 cctgtacggc atggacgagc tgtacaagag cggcctgaga agcatggtat ggcggccctt
     1861 ccatgatccc cgcctctccc agaagccctg actcctcctg ctttgcgccg tgcttttcct
     1921 ctgtagctcc cttgcttccc ccagcctcgg gtgtgggtgt ctaggccggg gttctggggc
     1981 aggcctgccg cgctcacccg tctgtctgct tgtctccctc tacagcctgg tccgaccccc
     2041 agtggcacta acgtgggatc ctcagggcgc tctcccagca aagcagtggc cgcccgggcg
     2101 gcgggatcca ctgtccggca gaggtaagga accctgcagt tcgttcgctt ccagactcgg
     2161 agataggacc cagaacctcg ctgattctgg ggtggagacc ctagcatgtg aagattgaca
     2221 aaggcaaaat gagcttctag tgacgtggcc gtgggagtag ttaaaggcct tttgggagga
     2281 aggcgacatt ttttttctcg ttgctcagtt tagggcacta ctcttaaaaa aggaaagtta
     2341 acaaactgga atagagtcag agataacttt gagaaaaccg atgtcattaa actggtgtct
     2401 ctggacctga ggtttgcact cacatttcca tctggcggcc ccataagcaa tctgtcctac
     2461 agataactcg tcctacacaa aacttagtct cttttcagct cagctctctc actctcaatt
     2521 atatctcctt acttccatat ggcactgttg tacactcatt tactcagagc cagaaacgtc
     2581 agcgtcatct tggatttttc ttatgctctt tctctctcta gtcatatgcc agactttaaa
     2641 ctctgcttga aagctttctc ataagctctt tccttttccc tttctactgc tttgcatttg
     2701 ctacttaacc cttttcttca ggctgtttgc tttccagtcc atcgttcgct ctgctgttac
     2761 tcttctgcgt agtttctgtt acttgttgct gaacatccca atggcgcttt gaggaagatg
     2821 aactgagatg tgtcagggtg acttatttcc atcatcgtcc ttaggggagc ttgggtaggg
     2881 gcaaggcgtg tagctgggac ctaggtccag acccctggct ctgccactga acggctcagg
     2941 tgctttgggc agttactccc cggcctcact ttgcacgtgt gcttacctag tggagacaaa
     3001 agtacatacc tcggtagagc gcgcacgcct gtaaccccag gttaactctc taacctcgag
     3061 ggggggcccg gtaccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa
     3121 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3181 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3241 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3301 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3361 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3421 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3481 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3541 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3601 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3661 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3721 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3781 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3841 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3901 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3961 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4021 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4081 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4141 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4201 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4261 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4321 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4381 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4441 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4501 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4561 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4621 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4681 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4741 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4801 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4861 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4921 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4981 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5041 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5101 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5161 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5221 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctacg
     5281 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     5341 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5401 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5461 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5521 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5581 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5641 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5701 cgaattttaa caaaatatta acgcttacaa tttcccattc gccattcagg ctgcgcaact
     5761 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     5821 gtgctgcaag
//
