LOCUS       sequence-470412-        8114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor to integrate a 3xFLAG-HaloTag at the 
            endogenous ATG16L1 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8114)
  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 8114)
  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-ATG16L1 HRD
FEATURES             Location/Qualifiers
     source          1..8114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3524..3529,3530..3546,3547..3552))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4060..4108
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4133..4152
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(4244..4353)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        4377..4468
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     4393..4410
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             5130..5195
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    5196..5229
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    5236..5284
                     /note="synthetic polyadenylation signal"
     CDS             complement(5285..5884)
                     /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(5368..5388)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5865..5884
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(5891..6207)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5905..6040
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    6214..6247
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             6256..7146
                     /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             7159..7179
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     polyA_signal    7604..7738
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7641..7660)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7695..7714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7767..7932
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca
     3361 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa
     3421 acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
     3481 ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca
     3541 ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac
     3601 cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc
     3661 ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg
     3721 cacggatctg cccttgcttc aggagatcgg tagacctcgg ccgtcgcggc gcttgccggt
     3781 ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt
     3841 gttcgcccag gactctagct atagttctag tggttggcta cgtacccgta gtggctatgg
     3901 cagggcttgc cgccccgacg ttggctgcga gccctgggcc ttcacccgaa cttgggggtt
     3961 ggggtgggga aaaggaagaa acgcgggcgt attggtccca atggggtctc ggtggggtat
     4021 cgacagagtg ccagccctgg gaccgaaccc cgcgtttatg aacaaacgac ccaacacccg
     4081 tgcgttttat tctgtctttt tattgccgtc atagcgcggg ttccttccgg tattgtctcc
     4141 ttccgtgttt cagttagcct cccccatctc ccggtaccgc atgctatgca tcagctgcta
     4201 gcaccatggc tcgagatccc gggtgatcaa gtcttcgtcg agtgattgta aataaaatgt
     4261 aatttacagt atagtatttt aattaatata caaatgattt gataataatt cttatttaac
     4321 tataatatat tgtgttgggt tgaattaaag gtccgtatac tccggaatat taatagatca
     4381 tggagataat taaaatgata accatctcgc aaataaataa gtattttact gttttcgtaa
     4441 cagttttgta ataaaaaaac ctataaatat tccggattat tcataccgtc ccaccatcgg
     4501 gcgcggatcc cggtccgaag cgcgcggaat tcaaaggcct acgtcgacga gctcactagt
     4561 cgcggccgct ttcgaatcta gagcctgcag tctcgacaag cttgtcgaga agtactagag
     4621 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     4681 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttgcag caaaagaaaa
     4741 gtaagcagtc aggcggaaac ggcccctttc ctgcctcctt cccgcccggc gctgggcctt
     4801 gcgcagggcc cggacgcccc caagcggcca cctctctgca gctgacgctt ccggctacag
     4861 gctggcgtca ctagcgagcg ccctgcgtag gcaccggctc ctgagcccgt gcttcgggtg
     4921 agggggcggg tcttccggcc ctctcgaaaa tcatttccgg catgagccgg aagaccgtcc
     4981 cggatggcct cggggactgc cagtgtgtgg aggtgagctc cgggattgcc ggcattcccg
     5041 cttctgctgg ttgcttcatg ctgcaggctg cggccgtcag ccctcgctcg cattggtggc
     5101 gctgaggtgc cggggcagca agtgacatgg actacaaaga ccatgacggt gattataaag
     5161 atcatgatat cgattacaag gatgacgatg acaagataac ttcgtataat gtatgctata
     5221 cgaagttata agcttcacac aaaaaaccaa cacacagatg taatgaaaat aaagatattt
     5281 tatttcaggc accgggcttg cgggtcatgc accaggtgcg cggtccttcg ggcacctcga
     5341 cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg
     5401 tctccaggaa ggcgggcacc ccggcgcgct cggccgcctc cactccgggg agcacgacgg
     5461 cgctgcccag acccttgccc tggtggtcgg gcgagacgcc gacggtggcc aggaaccacg
     5521 cgggctcctt gggccggtgc ggcgccagga ggccttccat ctgttgctgc gcggccagcc
     5581 gggaaccgct caactcggcc atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga
     5641 cgctctccgg cgtggtccag accgccaccg cggcgccgtc gtccgcgacc cacaccttgc
     5701 cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt
     5761 ggcggtccgg atcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg
     5821 tgcgtacggc cctggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg
     5881 tcatgagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata
     5941 gctcagaggc cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg
     6001 gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga
     6061 ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     6121 gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     6181 ctggggagcc tggggacttt ccacaccgga tccataactt cgtataatgt atgctatacg
     6241 aagttatccg gtaccatggc agaaatcggt actggctttc cattcgaccc ccattatgtg
     6301 gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg
     6361 ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt
     6421 gcaccgaccc atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca
     6481 gacctgggtt atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg
     6541 ggtctggaag aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg
     6601 gccaagcgca atccagagcg cgtcaaaggt attgcattta tggagttcat ccgccctatc
     6661 ccgacctggg acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc
     6721 gacgtcggcc gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg
     6781 ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat
     6841 cctgttgacc gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca
     6901 gcgaacatcg tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg
     6961 aagctgctgt tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg
     7021 gccaaaagcc tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa
     7081 gaagacaacc cggacctgat cggcagcgag atcgcgcgct ggctgtcgac gctcgagatt
     7141 tccggcgagc caaccactga ggatctgtac tttcagagcg ataacgcgat cgcttccggt
     7201 acctcgtcgc ccctccgcgc cgctgacttc ccccgctgga agcgccacat ctcggagcaa
     7261 ctgaggcgcc gggaccggct gcagagacag gcgttcgagg agatcatcct gcagtgtgag
     7321 cggcgccggt gcgggctggg agtggggcgg gcgggccccg cggaggcatg cggggcgtca
     7381 gcgggaacct gaggccgagt ccctggcgcc gcccgcacgc atggcggccg ccccgggtaa
     7441 cccgcgcttg ggacgccgca cgccttttaa atgttttttc taacctgcgt tttatctttg
     7501 gaggattttc cctattaaat tgtacgatct ttgtggggag gcacagttcg tgtttgtatt
     7561 tgtaaatccc agtagctcat acaatgctta tgcacttaaa tagaacttgt ttattgcagc
     7621 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     7681 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg
     7741 atcactgctt gagcctagga gatccgaacc agataagtga aatctagttc caaactattt
     7801 tgtcattttt aattttcgta ttagcttacg acgctacacc cagttcccat ctattttgtc
     7861 actcttccct aaataatcct taaaaactcc atttccaccc ctcccagttc ccaactattt
     7921 tgtccgccca cagcggggca tttttcttcc tgttatgttt ttaatcaaac atcctgccaa
     7981 ctccatgtga caaaccgtca tcttcggcta ctttttctct gtcacagaat gaaaattttt
     8041 ctgtcatctc ttcgttatta atgtttgtaa ttgactgaat atcaacgctt atttgcagcc
     8101 tgaatggcga atgg
//
