LOCUS       sequence-426858-        6159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template to tag endogenous human SEC61B N-terminus with 
            BSD.P2A.miniIAA7.3xFlag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6159)
  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 6159)
  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-SEC61B.N-BSD.P2A.miniIAA7.3xFlag
FEATURES             Location/Qualifiers
     source          1..6159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1451..1507
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1712..1777
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3491..3508)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3662..4250)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3742..3761)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4421..5212,5213..5281))
                     /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     5044..5063
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5282..5386)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5413..5868
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5500..5519)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5710..5731
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5999..6047
                     /note="synthetic polyadenylation signal"
     misc_feature    6061..6152
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6101..6120
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgagc tcttacgcgt aaccccagca atctgcaact ttaaatgggc ccacactaaa
       61 gttagagaac cacaggctcg ctcacaaccc tgacttctcc atgtcagttc cgatctttgc
      121 gaaccgcaga cagggaaggt cttctctcag gggtcatgcc cgcggccgcc ctccacggcg
      181 aggtccgcac tcgcgcagcc ggccccgcgg ccgcctcacc tggtcgcaca ctaccacgtc
      241 gaactcctcg tcggcgagga acagcacgta gagcgccagg aaaaccatgc gcacgtaggc
      301 gcagacggcg gcgccgcggc cgccccagcc caggcctcgc ggcagccagt ccccggcaca
      361 gcgcaccggt agctcgcggc tctcggcgaa acagtggccc gggtcgtagt gcgctgtcca
      421 gatcttcacg ctacacccgc gcgcctgcag cgccagcgcc gcgtccaaca ccagccgctc
      481 agcgccgccc acgcccaggt ctgggtggag gaacagcacc gacggcttgg gaaccgagtc
      541 ccgttcccgg ccctgctcct ccgccatggc cctggagccg caactgcacc ccgcaccctg
      601 atgggggtct tctgcgcaag ctccgcgctc gtagctccca gctggccact gcgggccgac
      661 cccgccctgc cgtacgtgcg tcagttaggc cacatcagcg caaatctgtg agggtctagt
      721 aactgcctga gaaaatatct tgtctgaccc cggttatatt tttccttcgg tagggattgg
      781 actttctgaa ggacgttgtg atccaaagga aggaggccgg aggtctctac ttcccataca
      841 gcaggtaact aagttgtctg tagcagactg tctacaggca tatcgtgaga cgacccaggc
      901 gtccctgggg tcagagagga ccttgcctgc aagtccgggg gcggggcctg agtcagtctc
      961 gccagctgcc ggtctttcgg gggctccgta actttctatc cgtccgcgtc agcgccttgc
     1021 caccctcatc tccaatatgc ctaggggagc caagccactg agccaggagg agagcacact
     1081 gattgaaaga gcaactgcca ctattaacag cattcccatt agcgaggact attctgtggc
     1141 aagcgccgcc ctgagctccg acggaaggat cttcaccggc gtgaacgtgt accactttac
     1201 aggcggcccc tgcgccgagc tggtggtgct gggaaccgca gcagcagcag cagcaggcaa
     1261 tctgacatgt atcgtggcca tcggcaacga gaataggggc atcctgtccc catgcggccg
     1321 gtgtagacag gtgctgctgg atctgcaccc tggcatcaag gccatcgtga aggactctga
     1381 tggacagcca accgccgtcg ggattagaga actgctgcca tcaggatacg tctgggaggg
     1441 cggctccggc gcaaccaact tttctctgct gaagcaggcc ggcgacgtgg aagaaaatcc
     1501 aggccccggc ttctctgaga ccgtggacct gatgctgaac ctgcagtcca ataaggaggg
     1561 ctctgtggat ctgaagaacg tgagcgccgt gcctaaggag aagaccacac tgaaggaccc
     1621 atccaagccc cctgccaagg cacaggtggt gggatggcca cccgtgcgga actacagaaa
     1681 gaatatgatg acccagcaga agacaagctc cgactacaaa gaccatgacg gtgattataa
     1741 agatcatgac atcgattaca aggatgacga tgacaagtcc ggaggcggag ggagcggggg
     1801 aggcggatct ggcggaggcg gatccatggt atggcggccc ttccatgatc cccgcctctc
     1861 ccagaagccc tgactcctcc tgctttgcgc cgtgcttttc ctctgtagct cccttgcttc
     1921 ccccagcctc gggtgtgggt gtctaggccg gggttctggg gcaggcctgc cgcgctcacc
     1981 cgtctgtctg cttgtctccc tctacagcct ggtccgaccc ccagtggcac taacgtggga
     2041 tcctcagggc gctctcccag caaagcagtg gccgcccggg cggcgggatc cactgtccgg
     2101 cagaggtaag gaaccctgca gttcgttcgc ttccagactc ggagatagga cccagaacct
     2161 cgctgattct ggggtggaga ccctagcatg tgaagattga caaaggcaaa atgagcttct
     2221 agtgacgtgg ccgtgggagt agttaaaggc cttttgggag gaaggcgaca ttttttttct
     2281 cgttgctcag tttagggcac tactcttaaa aaaggaaagt taacaaactg gaatagagtc
     2341 agagataact ttgagaaaac cgatgtcatt aaactggtgt ctctggacct gaggtttgca
     2401 ctcacatttc catctggcgg ccccataagc aatctgtcct acagataact cgtcctacac
     2461 aaaacttagt ctcttttcag ctcagctctc tcactctcaa ttatatctcc ttacttccat
     2521 atggcactgt tgtacactca tttactcaga gccagaaacg tcagcgtcat cttggatttt
     2581 tcttatgctc tttctctctc tagtcatatg ccagacttta aactctgctt gaaagctttc
     2641 tcataagctc tttccttttc cctttctact gctttgcatt tgctacttaa cccttttctt
     2701 caggctgttt gctttccagt ccatcgttcg ctctgctgtt actcttctgc gtagtttctg
     2761 ttacttgttg ctgaactaaa aaaaacaaac aaacaaaaaa aaaaaaccgg ggggatacca
     2821 aagctcttag tggtcatttc agcctaacct ttctttttcc tttttttttt tttgggatgg
     2881 aatctcactc tgtcgcccag gctggagtgc agtggcacga tctcggctca ctacagcctc
     2941 tgcctccaga gttcaagcaa ttctcctgcc tcagcctcct gagtagctga gactgcaggc
     3001 acgtgccacc acacccagct aattttttgt atttttagta gagacggggt ttcaccatgt
     3061 tagccaggat ggtctcgatc tcctgacctt gtgatcctcc gtctctgcct ccaaaagtgc
     3121 tggaattaca ggcgtgagcc actgcgcccg gccttcagcc taatctttct ttgctgtcct
     3181 atgatccagc actcgtgaac ccattactgt tttctgaatt cacaaacttt ctccctcatc
     3241 tcaaggcaaa tttctactta ttttccaaga ctaagctcag gggttatttc ctcttgaggc
     3301 cttctctgtc tgtaagcagc agtagctact agttttccct tttgccccca gtcgaccgat
     3361 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     3421 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     3481 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3541 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3601 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3661 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3721 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3781 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3841 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3901 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3961 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4021 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4081 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4141 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4201 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4261 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4321 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4381 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4441 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4501 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4561 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4621 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4681 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4741 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4801 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4861 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4921 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4981 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5041 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5101 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5161 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5221 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5281 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5341 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5401 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5461 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5521 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5581 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5641 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5701 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5761 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5821 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttgc cattcgccat
     5881 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     5941 ccaagctacc atgataagta agtaatatta aggtacggga ggtacttgga gcggccgcaa
     6001 taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagtact
     6061 aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc
     6121 cagtgcaagt gcaggtgcca gaacatttct ctatcgata
//
