LOCUS       sequence-223539-        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     48..70
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     62..79
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     63..79
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     86..105
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        86..104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     137..153
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     137..153
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1145..1846
                     /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(1278..1296)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1546..1565
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1740..1763
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     promoter        complement(3105..3123)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3144..3160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3144..3160)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3157..3179)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3168..3184
                     /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(3192..3198,3199..3216,3217..3222))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3237..3258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3375..3392)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3546..4134)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3626..3645)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4305..5096,5097..5165))
                     /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     4928..4947
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5166..5270)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5296..5751)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5433..5454)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5645..5664
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
       61 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcca
      121 ccgcggtggc ggccgctcta gaactagtgg atccgggccc acactaaagt tagagaacca
      181 caggctcgct cacaaccctg acttctccat gtcagttccg atctttgcga accgcagaca
      241 gggaaggtct tctctcaggg gtcatgcccg cggccgccct ccacggcgag gtccgcactc
      301 gcgcagccgg ccccgcggcc gcctcacctg gtcgcacact accacgtcga actcctcgtc
      361 ggcgaggaac agcacgtaga gcgccaggaa aaccatgcgc acgtaggcgc agacggcggc
      421 gccgcggccg ccccagccca ggcctcgcgg cagccagtcc ccggcacagc gcaccggtag
      481 ctcgcggctc tcggcgaaac agtggcccgg gtcgtagtgc gctgtccaga tcttcacgct
      541 acacccgcgc gcctgcagcg ccagcgccgc gtccaacacc agccgctcag cgccgcccac
      601 gcccaggtct gggtggagga acagcaccga cggcttggga accgagtccc gttcccggcc
      661 ctgctcctcc gccatggccc tggagccgca actgcacccc gcaccctgat gggggtcttc
      721 tgcgcaagct ccgcgctcgt agctcccagc tggccactgc gggccgaccc cgccctgccg
      781 tacgtgcgtc agttaggcca catcagcgca aatctgtgag ggtctagtaa ctgcctgaga
      841 aaatatcttg tctgaccccg gttatatttt tccttcggta gggattggac tttctgaagg
      901 acgttgtgat ccaaaggaag gaggccggag gtctctactt cccatacagc aggtaactaa
      961 gttgtctgta gcagactgtc tacaggcata tcgtgagacg acccaggcgt ccctggggtc
     1021 agagaggacc ttgcctgcaa gtccgggggc ggggcctgag tcagtctcgc cagctgccgg
     1081 tctttcgggg gctccgtaac tttctatccg tccgcgtcag cgccttgcca cactcatctc
     1141 caatatggtg agcaagggcg aggaggtcat caaagagttc atgcgcttca aggtgcgcat
     1201 ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1261 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     1321 ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc accccgccga
     1381 catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     1441 cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg acggcacgct
     1501 gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg taatgcagaa
     1561 gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa
     1621 gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa
     1681 gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg tggacaccaa
     1741 gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgctccga
     1801 gggccgccac cacctgttcc tgtacggcat ggacgagctg tacaagagcg gcctgagaag
     1861 catggtatgg cggcccttcc atgatccccg cctctcccag aagccctgac tcctcctgct
     1921 ttgcgccgtg cttttcctct gtagctccct tgcttccccc agcctcgggt gtgggtgtct
     1981 aggccggggt tctggggcag gcctgccgcg ctcacccgtc tgtctgcttg tctccctcta
     2041 cagcctggtc cgacccccag tggcactaac gtgggatcct cagggcgctc tcccagcaaa
     2101 gcagtggccg cccgggcggc gggatccact gtccggcaga ggtaaggaac cctgcagttc
     2161 gttcgcttcc agactcggag ataggaccca gaacctcgct gattctgggg tggagaccct
     2221 agcatgtgaa gattgacaaa ggcaaaatga gcttctagtg acgtggccgt gggagtagtt
     2281 aaaggccttt tgggaggaag gcgacatttt ttttctcgtt gctcagttta gggcactact
     2341 cttaaaaaag gaaagttaac aaactggaat agagtcagag ataactttga gaaaaccgat
     2401 gtcattaaac tggtgtctct ggacctgagg tttgcactca catttccatc tggcggcccc
     2461 ataagcaatc tgtcctacag ataactcgtc ctacacaaaa cttagtctct tttcagctca
     2521 gctctctcac tctcaattat atctccttac ttccatatgg cactgttgta cactcattta
     2581 ctcagagcca gaaacgtcag cgtcatcttg gatttttctt atgctctttc tctctctagt
     2641 catatgccag actttaaact ctgcttgaaa gctttctcat aagctctttc cttttccctt
     2701 tctactgctt tgcatttgct acttaaccct tttcttcagg ctgtttgctt tccagtccat
     2761 cgttcgctct gctgttactc ttctgcgtag tttctgttac ttgttgctga acatcccaat
     2821 ggcgctttga ggaagatgaa ctgagatgtg tcagggtgac ttatttccat catcgtcctt
     2881 aggggagctt gggtaggggc aaggcgtgta gctgggacct aggtccagac ccctggctct
     2941 gccactgaac ggctcaggtg ctttgggcag ttactccccg gcctcacttt gcacgtgtgc
     3001 ttacctagtg gagacaaaag tacatacctc ggtagagcgc gcacgcctgt aaccccaggt
     3061 taactctcta acctcgaggg ggggcccggt accagctttt gttcccttta gtgagggtta
     3121 atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3181 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3241 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3301 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3361 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3421 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3481 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3541 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3601 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3661 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3721 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3781 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3841 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3901 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3961 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4021 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4081 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4141 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4201 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4261 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4321 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4381 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4441 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4501 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4561 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4621 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4681 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4741 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4801 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4861 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4921 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4981 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5041 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5101 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5161 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5221 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5281 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     5341 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     5401 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     5461 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     5521 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     5581 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     5641 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     5701 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     5761 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     5821 agctggcgaa
//
