LOCUS       sequence-426852-        4133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express i53 in mammalian cells as a HDR enhancer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4133)
  AUTHORS   Li S, Wang Y, van der Stoel M, Zhou X, Madhusudan S, Kanerva K, 
            Nguyen VD, Eskici N, Olkkonen VM, Zhou Y, Raivio T, Ikonen E
  TITLE     HiHo-AID2: boosting homozygous knock-in efficiency enables robust 
            generation of human auxin-inducible degron cells.
  JOURNAL   Genome Biol. 2024 Feb 26;25(1):58. doi: 10.1186/s13059-024-03187-w.
  PUBMED    38409044
REFERENCE   2  (bases 1 to 4133)
  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"; journalName: "Genome 
            Biol."; date: "2024-02-26"; volume: "25(1):58. doi"; pages: " 
            10.1186/s13059-024-03187-w"
COMMENT     Sequence Label: pGL3-EF1a-i53
FEATURES             Location/Qualifiers
     source          1..4133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..31)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(185..773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(265..284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(944..1735,1736..1804))
                     /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     1567..1586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1805..1909)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1977..3139
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2208..3130
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3087..3107
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     intron          3178..3310
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3293..3312
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        3355..3373
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3399..3422
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3873..3892)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3917..3964
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
       61 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      121 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      181 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      241 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      301 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      361 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
      421 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
      481 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      541 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      601 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
      661 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      721 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      781 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
      841 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
      901 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
      961 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     1021 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     1081 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     1141 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     1201 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     1261 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     1321 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     1381 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     1441 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     1501 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     1561 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     1621 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     1681 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     1741 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     1801 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     1861 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1921 gaaaagtgcc acctgacggt accgctagcg tcgacggatc tcgagtctag tcgtgaggct
     1981 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     2041 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     2101 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2161 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt
     2221 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt
     2281 ccacctggct ccagtacgtg attcttgatc ccgagctgga gccaggggcg ggccttgcgc
     2341 tttaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
     2401 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     2461 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     2521 gggccaggat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     2581 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     2641 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     2701 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     2761 gccgcttccc ggccctgctc cagggggctc aaaatggagg acgcggcgct cgggagagcg
     2821 ggcgggtgag tcacccacac aaaggaaagg ggcctttccg tcctcagccg tcgcttcatg
     2881 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctgga gcttttggag
     2941 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     3001 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     3061 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     3121 tccatttcag gtgtcgtgaa cacgggctgc agaagttggt cgtgaggcac tgggcaggta
     3181 agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag
     3241 agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt
     3301 ctctccacag gtgtccactc ccagttcaat tacagctctt aaggctagag tacttaatac
     3361 gactcactat aggctagcct cgagaattca ccatgggaga ctacaaggat gacgatgaca
     3421 agggatctgc cggcagtgct gccggaagcg gcgagttcct gatcttcgtg aagacactga
     3481 ctggcaagac catcacactg gaagtggagc cttccgacac cattgagaac gtgaaggcta
     3541 agattcagga caaggagggc attcctccag accagcagag gctggccttc gctggcaagt
     3601 ctctggagga tggaaggact ctgtctgact acaacatcct gaaggactcc aaactgcacc
     3661 ccctgctgcg gctgcgctga tcgatgcggg actctggggt tcgaaatgac cgaccaagcg
     3721 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3781 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3841 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     3901 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     3961 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     4021 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     4081 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctc
//
