LOCUS       sequence-223489-        5845 bp    DNA     circular SYN 15-APR-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 Apr 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"
     CDS             join(924..926,927..929,930..1640)
                     /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(969..990)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1230..1249)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1577..1598
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2899..2917)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2938..2954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2938..2954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2951..2973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2962..2978
                     /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(2986..2992,2993..3010,3011..3016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3031..3052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3169..3186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3340..3928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3420..3439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4099..4890,4891..4959))
                     /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     4722..4741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4960..5064)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5090..5545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5177..5196)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5387..5408
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5672..5694
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5686..5703
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5687..5703
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5710..5729
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5710..5728
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5761..5777
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5761..5777
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 tctttgcgaa ccgcagacag ggaaggtctt ctctcagggg tcatgcccgc ggccgccctc
       61 cacggcgagg tccgcactcg cgcagccggc cccgcggccg cctcacctgg tcgcacacta
      121 ccacgtcgaa ctcctcgtcg gcgaggaaca gcacgtagag cgccaggaaa accatgcgca
      181 cgtaggcgca gacggcggcg ccgcggccgc cccagcccag gcctcgcggc agccagtccc
      241 cggcacagcg caccggtagc tcgcggctct cggcgaaaca gtggcccggg tcgtagtgcg
      301 ctgtccagat cttcacgcta cacccgcgcg cctgcagcgc cagcgccgcg tccaacacca
      361 gccgctcagc gccgcccacg cccaggtctg ggtggaggaa cagcaccgac ggcttgggaa
      421 ccgagtcccg ttcccggccc tgctcctccg ccatggccct ggagccgcaa ctgcaccccg
      481 caccctgatg ggggtcttct gcgcaagctc cgcgctcgta gctcccagct ggccactgcg
      541 ggccgacccc gccctgccgt acgtgcgtca gttaggccac atcagcgcaa atctgtgagg
      601 gtctagtaac tgcctgagaa aatatcttgt ctgaccccgg ttatattttt ccttcggtag
      661 ggattggact ttctgaagga cgttgtgatc caaaggaagg aggccggagg tctctacttc
      721 ccatacagca ggtaactaag ttgtctgtag cagactgtct acaggcatat cgtgagacga
      781 cccaggcgtc cctggggtca gagaggacct tgcctgcaag tccgggggcg gggcctgagt
      841 cagtctcgcc agctgccggt ctttcggggg ctccgtaact ttctatccgt ccgcgtcagc
      901 gccttgccac actcatctcc aatatggtga gcaagggcga ggagctgttc accggggtgg
      961 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1021 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1081 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1141 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1201 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1261 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1321 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1381 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1441 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1501 ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca
     1561 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1621 gcatggacga gctgtacaag agcggcctga gaagcatggt atggcggccc ttccatgatc
     1681 cccgcctctc ccagaagccc tgactcctcc tgctttgcgc cgtgcttttc ctctgtagct
     1741 cccttgcttc ccccagcctc gggtgtgggt gtctaggccg gggttctggg gcaggcctgc
     1801 cgcgctcacc cgtctgtctg cttgtctccc tctacagcct ggtccgaccc ccagtggcac
     1861 taacgtggga tcctcagggc gctctcccag caaagcagtg gccgcccggg cggcgggatc
     1921 cactgtccgg cagaggtaag gaaccctgca gttcgttcgc ttccagactc ggagatagga
     1981 cccagaacct cgctgattct ggggtggaga ccctagcatg tgaagattga caaaggcaaa
     2041 atgagcttct agtgacgtgg ccgtgggagt agttaaaggc cttttgggag gaaggcgaca
     2101 ttttttttct cgttgctcag tttagggcac tactcttaaa aaaggaaagt taacaaactg
     2161 gaatagagtc agagataact ttgagaaaac cgatgtcatt aaactggtgt ctctggacct
     2221 gaggtttgca ctcacatttc catctggcgg ccccataagc aatctgtcct acagataact
     2281 cgtcctacac aaaacttagt ctcttttcag ctcagctctc tcactctcaa ttatatctcc
     2341 ttacttccat atggcactgt tgtacactca tttactcaga gccagaaacg tcagcgtcat
     2401 cttggatttt tcttatgctc tttctctctc tagtcatatg ccagacttta aactctgctt
     2461 gaaagctttc tcataagctc tttccttttc cctttctact gctttgcatt tgctacttaa
     2521 cccttttctt caggctgttt gctttccagt ccatcgttcg ctctgctgtt actcttctgc
     2581 gtagtttctg ttacttgttg ctgaacatcc caatggcgct ttgaggaaga tgaactgaga
     2641 tgtgtcaggg tgacttattt ccatcatcgt ccttagggga gcttgggtag gggcaaggcg
     2701 tgtagctggg acctaggtcc agacccctgg ctctgccact gaacggctca ggtgctttgg
     2761 gcagttactc cccggcctca ctttgcacgt gtgcttacct agtggagaca aaagtacata
     2821 cctcggtaga gcgcgcacgc ctgtaacccc aggttaactc tctaacctcg agggggggcc
     2881 cggtaccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt aatcatggtc
     2941 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3001 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3061 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3121 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3181 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3241 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3301 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3361 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3421 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3481 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3541 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3601 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3661 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3721 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3781 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3841 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3901 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3961 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4021 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4081 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4141 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4201 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4261 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4321 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4381 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4441 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4501 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4561 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4621 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4681 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4741 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4801 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4861 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4921 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4981 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5041 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta cgcgccctgt
     5101 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5161 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5221 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5281 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5341 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5401 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5461 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5521 aacaaaatat taacgcttac aatttcccat tcgccattca ggctgcgcaa ctgttgggaa
     5581 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     5641 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     5701 agtgaattgt aatacgactc actatagggc gaattggagc tccaccgcgg tggcggccgc
     5761 tctagaacta gtggatccgg gcccacacta aagttagaga accacaggct cgctcacaac
     5821 cctgacttct ccatgtcagt tccga
//
