LOCUS       sequence-218836-        5845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor to insert mEGFP at the SEC61B locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5845)
  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 5845)
  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-mEGFP
FEATURES             Location/Qualifiers
     source          1..5845
                     /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             join(1224..1226,1227..1229,1230..1940)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1269..1290)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1530..1549)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1877..1898
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3199..3217)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3238..3254)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3238..3254)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3251..3273)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3262..3278
                     /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(3286..3292,3293..3310,3311..3316))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3331..3352
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3469..3486)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3640..4228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3720..3739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4399..5190,5191..5259))
                     /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     5022..5041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5260..5364)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5390..5818
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5477..5496)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5687..5708
                     /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 ggagctgttc accggggtgg
     1261 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1321 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1381 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1441 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1501 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1561 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1621 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1681 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1741 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1801 ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca
     1861 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1921 gcatggacga gctgtacaag agcggcctga gaagcatggt atggcggccc ttccatgatc
     1981 cccgcctctc ccagaagccc tgactcctcc tgctttgcgc cgtgcttttc ctctgtagct
     2041 cccttgcttc ccccagcctc gggtgtgggt gtctaggccg gggttctggg gcaggcctgc
     2101 cgcgctcacc cgtctgtctg cttgtctccc tctacagcct ggtccgaccc ccagtggcac
     2161 taacgtggga tcctcagggc gctctcccag caaagcagtg gccgcccggg cggcgggatc
     2221 cactgtccgg cagaggtaag gaaccctgca gttcgttcgc ttccagactc ggagatagga
     2281 cccagaacct cgctgattct ggggtggaga ccctagcatg tgaagattga caaaggcaaa
     2341 atgagcttct agtgacgtgg ccgtgggagt agttaaaggc cttttgggag gaaggcgaca
     2401 ttttttttct cgttgctcag tttagggcac tactcttaaa aaaggaaagt taacaaactg
     2461 gaatagagtc agagataact ttgagaaaac cgatgtcatt aaactggtgt ctctggacct
     2521 gaggtttgca ctcacatttc catctggcgg ccccataagc aatctgtcct acagataact
     2581 cgtcctacac aaaacttagt ctcttttcag ctcagctctc tcactctcaa ttatatctcc
     2641 ttacttccat atggcactgt tgtacactca tttactcaga gccagaaacg tcagcgtcat
     2701 cttggatttt tcttatgctc tttctctctc tagtcatatg ccagacttta aactctgctt
     2761 gaaagctttc tcataagctc tttccttttc cctttctact gctttgcatt tgctacttaa
     2821 cccttttctt caggctgttt gctttccagt ccatcgttcg ctctgctgtt actcttctgc
     2881 gtagtttctg ttacttgttg ctgaacatcc caatggcgct ttgaggaaga tgaactgaga
     2941 tgtgtcaggg tgacttattt ccatcatcgt ccttagggga gcttgggtag gggcaaggcg
     3001 tgtagctggg acctaggtcc agacccctgg ctctgccact gaacggctca ggtgctttgg
     3061 gcagttactc cccggcctca ctttgcacgt gtgcttacct agtggagaca aaagtacata
     3121 cctcggtaga gcgcgcacgc ctgtaacccc aggttaactc tctaacctcg agggggggcc
     3181 cggtaccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt aatcatggtc
     3241 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3301 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3361 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3421 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3481 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3541 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3601 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3661 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3721 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3781 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3841 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3901 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3961 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4021 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4081 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4141 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4201 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4261 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4321 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4381 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4441 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4501 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4561 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4621 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4681 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4741 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4801 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4861 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4921 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4981 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5041 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5101 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5161 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5221 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5281 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5341 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta cgcgccctgt
     5401 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5461 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5521 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5581 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5641 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5701 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5761 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5821 aacaaaatat taacgcttac aattt
//
