LOCUS       sequence-367849-        5737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to endogenously knock out the ANKRD1 in Hek293 cells. 
            eEF1a promoter, puromycin and polyA sequence is inserted between two
            homology arms. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5737)
  AUTHORS   Meng Q, Stoyko D, Andrews CM, Konstantinidou P, Genzor P, O T, 
            Elchert AR, Benner L, Sobti S, Katz EY, Haase AD
  TITLE     Functional editing of endogenous genes through rapid selection of 
            cell pools (Rapid generation of endogenously tagged genes in 
            Drosophila ovarian somatic sheath cells).
  JOURNAL   Nucleic Acids Res. 2022 May 27. pii: 6594084. doi: 
            10.1093/nar/gkac448.
  PUBMED    35639929
REFERENCE   2  (bases 1 to 5737)
  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: "Nucleic 
            Acids Res. 2022 May 27. pii: 6594084. doi: 10.1093/nar/gkac448."
COMMENT     Sequence Label: Donor_ANKRD1_KO_Puro
FEATURES             Location/Qualifiers
     source          1..5737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2..19)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(173..761)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(253..272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(932..1723,1724..1792))
                     /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     1555..1574
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1793..1897)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1965..1983
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2021..2043)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2143..2162
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2356..2378
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2370..2387
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2371..2387
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2915..3049
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2939..2958)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2993..3012
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3050..3649)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3133..3153)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3630..3649
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3684..4870)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3693..4639
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3716..3736)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(5508..5524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5508..5524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5521..5543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5532..5548
                     /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(5556..5562,5563..5580,5581..5586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5601..5622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
       61 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
      121 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
      181 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      241 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
      301 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
      361 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
      421 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
      481 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
      541 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
      601 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
      661 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
      721 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
      781 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
      841 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
      901 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
      961 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1021 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1081 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     1141 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     1201 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     1261 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     1321 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     1381 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     1441 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     1501 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     1561 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     1621 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     1681 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     1741 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     1801 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     1861 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     1921 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     1981 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     2041 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     2101 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     2161 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     2221 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     2281 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     2341 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg
     2401 gtacccgggg atcctctagg atgcttgtcc tactctcttg gttaaatcaa agcttgggtt
     2461 aggttgacag attaaatcta cttttaatct gcttaatcac atttctttta ggaaaatcaa
     2521 aaaccttctc tgcttaatta aaactcaact ttaggtactt ttgatgagtt tcgccttatt
     2581 gtaaagtttc cgggcttaca ttctttcaat aacacttctt ccctaagaca ttttgataac
     2641 tacacgtgat tccacactgt agtaataaaa ccaaaggaga agatattaaa taggaaatat
     2701 gcagagaatt cattactggt agctccattt tttttttcca atttcacttg tttcatcaca
     2761 tcaacctttt tccatatatg acattttcag agttcccttg cattacacct gaaagcaaag
     2821 ggcatttcaa ttttcaaata aaaaccaaga tgaaccagtt ttcattttat aaaattaatg
     2881 catcttacat accagttcct ctactttcag taccgatcca gacatgataa gatacattga
     2941 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     3001 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttc taggcaccgg
     3061 gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg gcggtgacgg
     3121 tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtctcc aggaaggcgg
     3181 gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg cccagaccct
     3241 tgccctggtg gtcgggcgag acgccgacgg tggccaggaa ccacgcgggc tccttgggcc
     3301 ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa ccgctcaact
     3361 cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc tccggcgtgg
     3421 tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg tcgagcccga
     3481 cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg tccgggtcga
     3541 cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt acggcccggg
     3601 ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcatt ttgattgcta
     3661 gcaagcttgg tcaagctaat tcctcacgac acctgaaatg gaagaaaaaa actttgaacc
     3721 actgtctgag gcttgagaat gaaccaagat ccaaactcaa aaagggcaaa ttccaaggag
     3781 aattacatca agtgccaagc tggcctaact tcagtctcca cccactcagt gtggggaaac
     3841 tccatcgcat aaaacccctc cccccaacct aaagacgacg tactccaaaa gctcgatcga
     3901 gaactaatcg aggtgcctgg acggcgcccg gtactccgtg gagtcacatg aagcgacggc
     3961 tgaggacgga aaggcccttt tcctttgtgt gggtgactca cccgcccgct ctcccgagcg
     4021 ccgcgtcctc cattttgagc tccctgcagc agggccggga agcggccatc tttccgctca
     4081 cgcaactggt gccgaccggg ccagccttgc cgcccagggc ggggcgatac acggcggcgc
     4141 gaggccaggc accagagcag gccggccagc ttgagactac ccccgtccga ttctcggtgg
     4201 ccgcgctcgc aggccccgcc tcgccgaaca tgtgcgctgg gacgcacggg ccccgtcgcc
     4261 gcccgcggcc ccaaaaaccg aaataccagt gtgcagatcg atcttggccc gcatttacaa
     4321 gactatcttg ccagaaaaaa agcgtcgcag caggtcatca aaaattttaa atggctagag
     4381 acttatcgaa agcagcgaga caggcgcgaa ggtgccacca gattcgcacg cggcggcccc
     4441 agcgcccagg ccaggcctca actcaagcac gaggcgaagg ggctccttaa gcgcaaggcc
     4501 tcgaactctc ccacccactt ccaacccgaa gctcgggatc aagaatcacg tactgcagcc
     4561 aggtggaagt aattcaaggc acgcaagggc cataacccgt aaagaggcca ggcccgcggg
     4621 aaccacacac ggcacttacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca
     4681 cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt
     4741 acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg gttcaattgc
     4801 cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc cactgacggg
     4861 caccggagcc aattcccact cctttcaaga cctagaaggt ccattagctg caaagattcc
     4921 tctctgttta aaactttatc catctttgca aagctcggtc gagatctccc gatccgtcga
     4981 gacgcgtagt acgttggctg aaggagtctt gtatgttttt ctgtcgtgga aggaatccct
     5041 ggagttggcc ctgctgggcc cctctacacc ggtcagctta tatagctgtt gccgggtgag
     5101 ataatcttcc aacctgggaa ccgaagtaac actcccaccc ccattaggag ccaactcaga
     5161 ggcaggtgaa ttttcattcc agactcagtg tctgggaagc tgaagaggga ggggaggaca
     5221 agctaaccct gctgaatatg tgaatcagga agaaatgtga gagggccatt cctttggtgg
     5281 tgatcacatc gctcagcaat gcaagtcatc ctatttgtca agagtcaggg gacagctgtc
     5341 tgttgacatt gcaccacatc actgcccttt ttttctttgt cagctttcat atgactacct
     5401 atcaagaaaa tgtagatgcc ctacatatca cacccccagt aatatctttc tgataagcag
     5461 acttatcaac acttcactta ggggaaactt gtcatggcgt aatcatggtc atagctgttt
     5521 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5581 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5641 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5701 gggagaggcg gtttgcgtat tgggcgctct tccgctt
//
