LOCUS       sequence-430513-        7099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor to integrate a 3xFLAG-HaloTag at the 
            endogenous ATG5 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7099)
  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 7099)
  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: Halo-ATG5 HRD
FEATURES             Location/Qualifiers
     source          1..7099
                     /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="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1399..1404,1405..1421,1422..1427))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    1935..1983
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2008..2027
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(2119..2228)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2252..2343
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2268..2285
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             3063..3128
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3135..4025
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             4038..4058
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     polyA_signal    4483..4617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4520..4539)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4574..4593
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  4646..4811
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      4995..5449
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5082..5101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5291..5312
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5475..5579
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5580..5648,5649..6440)
                     /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(5798..5817)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6611..7099,1..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 ccataacatc aaacatcgac ccacggcgta acgcgcttgc
     1261 tgcttggatg cccgaggcat agactgtaca aaaaaacagt cataacaagc catgaaaacc
     1321 gccactgcgc cgttaccacc gctgcgttcg gtcaaggttc tggaccagtt gcgtgagcgc
     1381 atacgctact tgcattacag tttacgaacc gaacaggctt atgtcaactg ggttcgtgcc
     1441 ttcatccgtt tccacggtgt gcgtcacccg gcaaccttgg gcagcagcga agtcgaggca
     1501 tttctgtcct ggctggcgaa cgagcgcaag gtttcggtct ccacgcatcg tcaggcattg
     1561 gcggccttgc tgttcttcta cggcaaggtg ctgtgcacgg atctgccctt gcttcaggag
     1621 atcggtagac ctcggccgtc gcggcgcttg ccggtggtgc tgaccccgga tgaagtggtt
     1681 cgcatcctcg gttttctgga aggcgagcat cgtttgttcg cccaggactc tagctatagt
     1741 tctagtggtt ggctacgtac ccgtagtggc tatggcaggg cttgccgccc cgacgttggc
     1801 tgcgagccct gggccttcac ccgaacttgg gggttggggt ggggaaaagg aagaaacgcg
     1861 ggcgtattgg tcccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg
     1921 aaccccgcgt ttatgaacaa acgacccaac acccgtgcgt tttattctgt ctttttattg
     1981 ccgtcatagc gcgggttcct tccggtattg tctccttccg tgtttcagtt agcctccccc
     2041 atctcccggt accgcatgct atgcatcagc tgctagcacc atggctcgag atcccgggtg
     2101 atcaagtctt cgtcgagtga ttgtaaataa aatgtaattt acagtatagt attttaatta
     2161 atatacaaat gatttgataa taattcttat ttaactataa tatattgtgt tgggttgaat
     2221 taaaggtccg tatactccgg aatattaata gatcatggag ataattaaaa tgataaccat
     2281 ctcgcaaata aataagtatt ttactgtttt cgtaacagtt ttgtaataaa aaaacctata
     2341 aatattccgg attattcata ccgtcccacc atcgggcgcg gatcccggtc cgaagcgcgc
     2401 ggaattcaaa ggcctacgtc gacgagctca ctagtcgcgg ccgctttcga atctagagcc
     2461 tgcagtctcg acaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt
     2521 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2581 atgaatgcaa ttgttgttgt tggggcaggg gtaaatgcga gaatgatcct ttggagttgg
     2641 actgtcagga ttcacagggt ataggtgaag ctcctcttta ggtaagcagt agactcttct
     2701 gggcttgaaa gactgatgca gatacagttt cattttgact gtggttcagt acttaaatat
     2761 gcaatgaata ttactggcat atgtaatttt gatctgattc ctttacataa gtaataattt
     2821 tacatttaga atttgttgta ctcttttctt tgaatttata ctaaagattg gtgatcatcg
     2881 tttttttttt tttgaagtgg ataatcttta tggcatggat tttttaaaat aatcttttgt
     2941 tttctagcag gttcattcat cgttgcaagg atctgactaa tgctcttcat tttattttca
     3001 gtttggcttt ggttgaaata agaatttagc ctgtatgtac tgctttaact cctggaagaa
     3061 tggactacaa agaccatgac ggtgattata aagatcatga tatcgattac aaggatgacg
     3121 atgacaaggg ttccatggca gaaatcggta ctggctttcc attcgacccc cattatgtgg
     3181 aagtcctggg cgagcgcatg cactacgtcg atgttggtcc gcgcgatggc acccctgtgc
     3241 tgttcctgca cggtaacccg acctcctcct acgtgtggcg caacatcatc ccgcatgttg
     3301 caccgaccca tcgctgcatt gctccagacc tgatcggtat gggcaaatcc gacaaaccag
     3361 acctgggtta tttcttcgac gaccacgtcc gcttcatgga tgccttcatc gaagccctgg
     3421 gtctggaaga ggtcgtcctg gtcattcacg actggggctc cgctctgggt ttccactggg
     3481 ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat ggagttcatc cgccctatcc
     3541 cgacctggga cgaatggcca gaatttgccc gcgagacctt ccaggccttc cgcaccaccg
     3601 acgtcggccg caagctgatc atcgatcaga acgtttttat cgagggtacg ctgccgatgg
     3661 gtgtcgtccg cccgctgact gaagtcgaga tggaccatta ccgcgagccg ttcctgaatc
     3721 ctgttgaccg cgagccactg tggcgcttcc caaacgagct gccaatcgcc ggtgagccag
     3781 cgaacatcgt cgcgctggtc gaagaataca tggactggct gcaccagtcc cctgtcccga
     3841 agctgctgtt ctggggcacc ccaggcgttc tgatcccacc ggccgaagcc gctcgcctgg
     3901 ccaaaagcct gcctaactgc aaggctgtgg acatcggccc gggtctgaat ctgctgcaag
     3961 aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg gctgtcgacg ctcgagattt
     4021 ccggcgagcc aaccactgag gatctgtact ttcagagcga taacgcgatc gcttccggta
     4081 ccacagatga caaagatgtg cttcgagatg tgtggtttgg acgaattcca acttgtttca
     4141 cgctatatca agatgagata actgaaaggg aagcagaacc atactatgta agtacattga
     4201 ttgtggcaaa tgggatattt aaccttgtcc atttttgtaa gaaaaagctg taagaacttg
     4261 gaggccatta tgtaacgttt tagatacagc taattcatgc ttattttgga aaaaaaatta
     4321 acacagataa gcataaagaa ggaaataatc acctctaatg ctaccactca gaggacaaaa
     4381 ccactgtcag catgttgtca tgatgcgttc tggactttgt tttccttaca tgtatgtggg
     4441 tatgttaatg gaatcatcag ctatggtgcc ttcttgactt tcaacttgtt tattgcagct
     4501 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4561 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctga
     4621 tcactgcttg agcctaggag atccgaacca gataagtgaa atctagttcc aaactatttt
     4681 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca
     4741 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt
     4801 gtccgcccac agcggggcat ttttcttcct gttatgtttt taatcaaaca tcctgccaac
     4861 tccatgtgac aaaccgtcat cttcggctac tttttctctg tcacagaatg aaaatttttc
     4921 tgtcatctct tcgttattaa tgtttgtaat tgactgaata tcaacgctta tttgcagcct
     4981 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5041 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5101 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5161 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5221 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5281 gttcttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5341 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5401 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5461 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5521 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5581 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5641 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5701 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5761 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5821 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5881 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5941 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     6001 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6061 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6121 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6181 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6241 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6301 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6361 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6421 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6481 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6541 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6601 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6661 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6721 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6781 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6841 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6901 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6961 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     7021 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7081 gcacgaggga gcttccagg
//
