LOCUS       sequence-430489-        8419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor to integrate a 3xFLAG-HaloTag at the 
            C-terminus of the endogenous ULK1 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8419)
  AUTHORS   Broadbent DG, Barnaba C, Perez GI, Schmidt JC
  TITLE     Quantitative analysis of autophagy reveals the role of ATG9 and ATG2
            in autophagosome formation.
  JOURNAL   J Cell Biol. 2023 Jul 3;222(7):e202210078. doi: 
            10.1083/jcb.202210078. Epub 2023 Apr 28.
  PUBMED    37115157
REFERENCE   2  (bases 1 to 8419)
  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: "J Cell 
            Biol."; date: "2023-07-3"; volume: "222(7):e202210078. doi"; pages: 
            " 10.1083/jcb.202210078. Epub 2023 Apr 28"
COMMENT     Sequence Label: ULK1-Halo HRD
FEATURES             Location/Qualifiers
     source          1..8419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     254..271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  405..629
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(696..1229)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1418..1423,1424..1440,1441..1446))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    1954..2002
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2027..2046
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(2138..2247)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2271..2362
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2287..2304
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             3092..3112
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             3128..4012
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             4013..4078
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    4111..4335
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4357..4686
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4537..4672
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4696..5295
                     /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(4696..4715)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5192..5212
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5296..5344
                     /note="synthetic polyadenylation signal"
     polyA_signal    5803..5937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5840..5859)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5894..5913
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  5966..6131
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      6315..6769
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6402..6421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6611..6632
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6795..6899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6900..6968,6969..7760)
                     /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(7118..7137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7931..8419,1..100)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
       61 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      121 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
      181 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
      241 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
      301 tctccttacg catctgtgcg gtatttcaca ccgcatagac cagccgcgta acctggcaaa
      361 atcggttacg gttgagtaat aaatggatgc cctgcgtaag cgggtgtggg cggacaataa
      421 agtcttaaac tgaacaaaat agatctaaac tatgacaata aagtcttaaa ctagacagaa
      481 tagttgtaaa ctgaaatcag tccagttatg ctgtgaaaaa gcatactgga cttttgttat
      541 ggctaaagca aactcttcat tttctgaagt gcaaattgcc cgtcgtatta aagaggggcg
      601 tggccaaggg catggtaaag actatattcg cggcgttgtg acaatttacc gaacaactcc
      661 gcggccggga agccgatctc ggcttgaacg aattgttagg tggcggtact tgggtcgata
      721 tcaaagtgca tcacttcttc ccgtatgccc aactttgtat agagagccac tgcgggatcg
      781 tcaccgtaat ctgcttgcac gtagatcaca taagcaccaa gcgcgttggc ctcatgcttg
      841 aggagattga tgagcgcggt ggcaatgccc tgcctccggt gctcgccgga gactgcgaga
      901 tcatagatat agatctcact acgcggctgc tcaaacttgg gcagaacgta agccgcgaga
      961 gcgccaacaa ccgcttcttg gtcgaaggca gcaagcgcga tgaatgtctt actacggagc
     1021 aagttcccga ggtaatcgga gtccggctga tgttgggagt aggtggctac gtctccgaac
     1081 tcacgaccga aaagatcaag agcagcccgc atggatttga cttggtcagg gccgagccta
     1141 catgtgcgaa tgatgcccat acttgagcca cctaactttg ttttagggcg actgccctgc
     1201 tgcgtaacat cgttgctgct gcgtaacatc gttgctgctc cataacatca aacatcgacc
     1261 cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata gactgtacaa aaaaacagtc
     1321 ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct
     1381 ggaccagttg cgtgagcgca tacgctactt gcattacagt ttacgaaccg aacaggctta
     1441 tgtcaactgg gttcgtgcct tcatccgttt ccacggtgtg cgtcacccgg caaccttggg
     1501 cagcagcgaa gtcgaggcat ttctgtcctg gctggcgaac gagcgcaagg tttcggtctc
     1561 cacgcatcgt caggcattgg cggccttgct gttcttctac ggcaaggtgc tgtgcacgga
     1621 tctgcccttg cttcaggaga tcggtagacc tcggccgtcg cggcgcttgc cggtggtgct
     1681 gaccccggat gaagtggttc gcatcctcgg ttttctggaa ggcgagcatc gtttgttcgc
     1741 ccaggactct agctatagtt ctagtggttg gctacgtacc cgtagtggct atggcagggc
     1801 ttgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg ggttggggtg
     1861 gggaaaagga agaaacgcgg gcgtattggt cccaatgggg tctcggtggg gtatcgacag
     1921 agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca cccgtgcgtt
     1981 ttattctgtc tttttattgc cgtcatagcg cgggttcctt ccggtattgt ctccttccgt
     2041 gtttcagtta gcctccccca tctcccggta ccgcatgcta tgcatcagct gctagcacca
     2101 tggctcgaga tcccgggtga tcaagtcttc gtcgagtgat tgtaaataaa atgtaattta
     2161 cagtatagta ttttaattaa tatacaaatg atttgataat aattcttatt taactataat
     2221 atattgtgtt gggttgaatt aaaggtccgt atactccgga atattaatag atcatggaga
     2281 taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt
     2341 tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg
     2401 atcccggtcc gaagcgcgcg gaattcaaag gcctacgtcg acgagctcac tagtcgcggc
     2461 cgctttcgaa tctagagcct gcagtctcga caagcttgtc gagaagtact agaggatcat
     2521 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2581 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt ccaggcagtg tctccccttg
     2641 gccctgtgac catcccctct gcacagcagg ctgcaccagc acttatgtct ccacgtcact
     2701 gccctgagcg cctatctgcc acccctgggc cgctgccgtg ggtgcagggc agcccttgct
     2761 gggagggagt ggggtgagct ggccctgtcc agcctctgtc ctcgccccca ggtgcagtcg
     2821 gctgccctgg acgagatgtt ccagcaccgt gagggctgcg tcccacgcta ccacaaggcc
     2881 ctgctgctcc tggaggggct gcagcacatg ctctcggacc aggccgacat cgagaacgtc
     2941 accaagtgtg agtgcccggc tgggctgggg gctggggact ctgggcgtct ccctcacact
     3001 cccctctccc tccacaggca agctgtgcat tgagcggaga ctctcggcgc tgctgactgg
     3061 catctgtgcc ggagctctcg agccaaccac tgaggatctg tactttcaga gcgataacga
     3121 tggatccgaa atcggtactg gctttccatt cgacccccat tatgtggaag tcctgggcga
     3181 gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg
     3241 taacccgacc tcctcctacg tgtggcgcaa catcatcccg catgttgcac cgacccatcg
     3301 ctgcattgct ccagacctga tcggtatggg caaatccgac aaaccagacc tgggttattt
     3361 cttcgacgac cacgtccgct tcatggatgc cttcatcgaa gccctgggtc tggaagaggt
     3421 cgtcctggtc attcacgact ggggctccgc tctgggtttc cactgggcca agcgcaatcc
     3481 agagcgcgtc aaaggtattg catttatgga gttcatccgc cctatcccga cctgggacga
     3541 atggccagaa tttgcccgcg agaccttcca ggccttccgc accaccgacg tcggccgcaa
     3601 gctgatcatc gatcagaacg tttttatcga gggtacgctg ccgatgggtg tcgtccgccc
     3661 gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg ttgaccgcga
     3721 gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga acatcgtcgc
     3781 gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc tgctgttctg
     3841 gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca aaagcctgcc
     3901 taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag acaacccgga
     3961 cctgatcggc agcgagatcg cgcgctggct gtctactctg gagatttccg gtgactacaa
     4021 agaccatgac ggtgattata aagatcatga tatcgattac aaggatgacg atgacaagta
     4081 actcgagtct agacgtttaa accctgcagg ctgtgccttc tagttgccag ccatctgttg
     4141 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     4201 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     4261 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     4321 cggtgggctc tatgggtcga cagtactaag ctttacgtgt gtcagttagg gtgtggaaag
     4381 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4441 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4501 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4561 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4621 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4681 tgcaaaaagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     4741 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     4801 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     4861 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     4921 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     4981 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     5041 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     5101 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     5161 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     5221 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     5281 aagcccggtg cctgaaataa aatatcttta ttttcattac atctgtgtgt tggttttttg
     5341 tgtgcctttc tggcctggct gggccccccg tcctgccgag ccctgcagag tgggctctgt
     5401 gtgctggctg gactcctcgg gacaagccca tggcgctgat cgctggtgct gagccctgcc
     5461 ctgggcccca cggacagtca gcctgccggc ctccctgcag ctcacggggc agaaccagca
     5521 catctggagc cacacagctt ggggggtgtc tcccatcttt tacaggtggg gatcacagaa
     5581 tttctgcccc tccagctgcc tggctcagca ggcgtgggtg ccaccaccct ctagccccag
     5641 ggcagccccg gaggacaggc aagggcctga gaccactgcc gactcaaagc caaagcgagc
     5701 tcctgcttag ggaaggtcag caggcactgt gcccaggaag agcctgcggc ctcggcgtcc
     5761 cccagtctcc aggagcctct ccctccgaga tacccaccca gcaacttgtt tattgcagct
     5821 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5881 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctga
     5941 tcactgcttg agcctaggag atccgaacca gataagtgaa atctagttcc aaactatttt
     6001 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca
     6061 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt
     6121 gtccgcccac agcggggcat ttttcttcct gttatgtttt taatcaaaca tcctgccaac
     6181 tccatgtgac aaaccgtcat cttcggctac tttttctctg tcacagaatg aaaatttttc
     6241 tgtcatctct tcgttattaa tgtttgtaat tgactgaata tcaacgctta tttgcagcct
     6301 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     6361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     6421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     6481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     6541 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     6601 gttcttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     6661 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     6721 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     6781 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     6841 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     6901 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     6961 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     7021 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     7081 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     7141 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     7201 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     7261 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     7321 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     7381 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     7441 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     7501 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     7561 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     7621 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     7681 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     7741 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     7801 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     7861 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     7921 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     7981 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     8041 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     8101 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     8161 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     8221 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     8281 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     8341 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     8401 gcacgaggga gcttccagg
//
