LOCUS       sequence-367854-        6163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to endogenously knock out the ANKRD1 in Hek293 cells. 
            eEF1a promoter, hygromycin 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 6163)
  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 6163)
  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_Hygro
FEATURES             Location/Qualifiers
     source          1..6163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     163..182
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     416..433
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    550..571
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(586..591,592..609,610..616)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    624..640
                     /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)."
     primer_bind     629..651
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     648..664
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     648..664
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1302..2488
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1533..2479
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2436..2456
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2523..3548
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2523..3548
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    3549..3683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3586..3605)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3640..3659
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4211..4228)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4211..4227)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4220..4242)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4436..4455)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4555..4577
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4615..4633)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4701..4805
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4806..4874,4875..5666)
                     /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     complement(5024..5043)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5837..6163,1..262)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
       61 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      121 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      181 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      241 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      301 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      361 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      421 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
      481 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      541 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
      601 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
      661 tgaccatgat tacgccatga caagtttccc ctaagtgaag tgttgataag tctgcttatc
      721 agaaagatat tactgggggt gtgatatgta gggcatctac attttcttga taggtagtca
      781 tatgaaagct gacaaagaaa aaaagggcag tgatgtggtg caatgtcaac agacagctgt
      841 cccctgactc ttgacaaata ggatgacttg cattgctgag cgatgtgatc accaccaaag
      901 gaatggccct ctcacatttc ttcctgattc acatattcag cagggttagc ttgtcctccc
      961 ctccctcttc agcttcccag acactgagtc tggaatgaaa attcacctgc ctctgagttg
     1021 gctcctaatg ggggtgggag tgttacttcg gttcccaggt tggaagatta tctcacccgg
     1081 caacagctat ataagctgac cggtgtagag gggcccagca gggccaactc cagggattcc
     1141 ttccacgaca gaaaaacata caagactcct tcagccaacg tactacgcgt ctcgacggat
     1201 cgggagatct cgaccgagct ttgcaaagat ggataaagtt ttaaacagag aggaatcttt
     1261 gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
     1321 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     1381 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     1441 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     1501 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
     1561 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
     1621 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
     1681 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
     1741 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     1801 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     1861 cgggccaaga tcgatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg
     1921 gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga
     1981 atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg
     2041 tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa
     2101 agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga
     2161 gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct
     2221 tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgatcgag
     2281 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     2341 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     2401 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     2461 tttttttctt ccatttcagg tgtcgtgagg aattagcttg accaagcttg ctagcaatca
     2521 aaatgaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     2581 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     2641 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     2701 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     2761 ttggggaatt tagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     2821 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     2881 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     2941 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     3001 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     3061 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     3121 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     3181 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     3241 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     3301 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     3361 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     3421 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     3481 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     3541 aggaatagaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3601 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3661 atgtatctta tcatgtctgg atcggtactg aaagtagagg aactggtatg taagatgcat
     3721 taattttata aaatgaaaac tggttcatct tggtttttat ttgaaaattg aaatgccctt
     3781 tgctttcagg tgtaatgcaa gggaactctg aaaatgtcat atatggaaaa aggttgatgt
     3841 gatgaaacaa gtgaaattgg aaaaaaaaaa tggagctacc agtaatgaat tctctgcata
     3901 tttcctattt aatatcttct cctttggttt tattactaca gtgtggaatc acgtgtagtt
     3961 atcaaaatgt cttagggaag aagtgttatt gaaagaatgt aagcccggaa actttacaat
     4021 aaggcgaaac tcatcaaaag tacctaaagt tgagttttaa ttaagcagag aaggtttttg
     4081 attttcctaa aagaaatgtg attaagcaga ttaaaagtag atttaatctg tcaacctaac
     4141 ccaagctttg atttaaccaa gagagtagga caagcatcct agaggatccc cgggtaccga
     4201 gctcgaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4261 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4321 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     4381 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     4441 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4501 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4561 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     4621 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4681 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4741 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4801 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4861 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4921 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4981 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5041 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5101 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5161 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5221 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5281 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5341 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5401 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5461 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5521 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5581 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5641 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5701 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5761 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5821 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5881 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5941 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6001 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6061 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6121 gatagttacc ggataaggcg cagcggtcgg gctgaacggg ggg
//
