LOCUS       sequence-429506-        7160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor to integrate a 3xFLAG-HaloTag at the 
            endogenous ATG2A locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7160)
  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 7160)
  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-ATG2A HRD
FEATURES             Location/Qualifiers
     source          1..7160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      102..556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(189..208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     398..419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        582..686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(687..755,756..1547)
                     /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(905..924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1718..2306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2207..2226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2460..2477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2611..2835
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2902..3435)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3605..3610,3611..3627,3628..3633))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    4141..4189
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4214..4233
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(4325..4434)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        4458..4549
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     4474..4491
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             5241..5306
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5313..6203
                     /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             6216..6236
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     RBS             6724..6732
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    6750..6884
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6787..6806)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6841..6860
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  6913..7078
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 ttctctgtca cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga
       61 ctgaatatca acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
      121 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      301 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      361 ttttcgccct ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga
      421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
      481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
      541 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      601 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
      661 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
      721 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
      781 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
      841 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
      901 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
      961 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1021 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1081 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1141 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1201 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     1261 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1321 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1381 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     1441 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1501 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     1561 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     1681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1861 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2101 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2161 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2221 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2281 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2341 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2401 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2461 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2521 tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg
     2581 agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa
     2641 caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga
     2701 aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact
     2761 cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg
     2821 gtaaagacta tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc
     2881 gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac
     2941 ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc
     3001 ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag
     3061 cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat
     3121 ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc
     3181 ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta
     3241 atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag
     3301 atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat
     3361 gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt
     3421 gctgctccat aacatcaaac atcgacccac ggcgtaacgc gcttgctgct tggatgcccg
     3481 aggcatagac tgtacaaaaa aacagtcata acaagccatg aaaaccgcca ctgcgccgtt
     3541 accaccgctg cgttcggtca aggttctgga ccagttgcgt gagcgcatac gctacttgca
     3601 ttacagttta cgaaccgaac aggcttatgt caactgggtt cgtgccttca tccgtttcca
     3661 cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc gaggcatttc tgtcctggct
     3721 ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag gcattggcgg ccttgctgtt
     3781 cttctacggc aaggtgctgt gcacggatct gcccttgctt caggagatcg gtagacctcg
     3841 gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa gtggttcgca tcctcggttt
     3901 tctggaaggc gagcatcgtt tgttcgccca ggactctagc tatagttcta gtggttggct
     3961 acgtacccgt agtggctatg gcagggcttg ccgccccgac gttggctgcg agccctgggc
     4021 cttcacccga acttgggggt tggggtgggg aaaaggaaga aacgcgggcg tattggtccc
     4081 aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc ccgcgtttat
     4141 gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg
     4201 gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtaccg
     4261 catgctatgc atcagctgct agcaccatgg ctcgagatcc cgggtgatca agtcttcgtc
     4321 gagtgattgt aaataaaatg taatttacag tatagtattt taattaatat acaaatgatt
     4381 tgataataat tcttatttaa ctataatata ttgtgttggg ttgaattaaa ggtccgtata
     4441 ctccggaata ttaatagatc atggagataa ttaaaatgat aaccatctcg caaataaata
     4501 agtattttac tgttttcgta acagttttgt aataaaaaaa cctataaata ttccggatta
     4561 ttcataccgt cccaccatcg ggcgcggatc ccggtccgaa gcgcgcggaa ttcaaaggcc
     4621 tacgtcgacg agctcactag tcgcggccgc tttcgaatct agagcctgca gtctcgacaa
     4681 gcttgtcgag aagtactaga ggatcataat cagccatacc acatttgtag aggttttact
     4741 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     4801 tgttgttgat gggacactgt ccctttaaat cctccccaac cacttaaagt ggttggacga
     4861 atcagcacat gcgcattcct ttgaggggcc ggcacctgga gcaacagcct tcccagagcc
     4921 tgtagagggg aggttaaagg tcctcccaca cagtacttta cgcaagcgta aatcatgatt
     4981 gagagcccta tatcatgcag agagcccagt ccctttttcg cgcccgaagg ctccgccccg
     5041 cgcagccaat cagtaggcct cgccctttgc aatagccaat gacagtgagc gtagggggcg
     5101 tggctgggcg tccgggggcg cggcctggcg cctagagaag cggcggggcg agccgggggc
     5161 tctagtgaac agcggagccg gacggggatc gccggcgggc ggcaagcgga ggcggcccag
     5221 gcccggcggt ctccgagatg gactacaaag accatgacgg tgattataaa gatcatgata
     5281 tcgattacaa ggatgacgat gacaagggtt ccatggcaga aatcggtact ggctttccat
     5341 tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc
     5401 gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca
     5461 acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg
     5521 gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg
     5581 ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg
     5641 ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg
     5701 agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc
     5761 aggccttccg caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg
     5821 agggtacgct gccgatgggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc
     5881 gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc
     5941 caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc
     6001 accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg
     6061 ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg
     6121 gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc
     6181 tgtcgacgct cgagatttcc ggcgagccaa ccactgagga tctgtacttt cagagcgata
     6241 acgcgatcgc ttccggtacc tcacgatggc tgtggccatg gtcaaactgt gtgaaagagc
     6301 gggtctgccg ctacttgctg caccactact taggtcactt cttccaagag cacctcagcc
     6361 tggaccagct cagcctcgat ctgtacaagg gcagcgttgc cctgcgagac atccacctgg
     6421 aaatctgggt gaggagccag gcccgagtcc aggaaggtgc ggagggtggg agcgcagcga
     6481 cctgaggctc cctgcggcag gatcgggctg ggggatcagg caccgggcgt agagaggcca
     6541 ggggaatctt gccctctccc aggcgtcaga ggcaccccgg ctagcctctt cagttcctta
     6601 ccagagccag aaactgagcg ggaaggggtt tctgtctcca cgggaggaga aactgaggct
     6661 ggagagggca agatcacctg tctggagaat gcaagacaaa ctcgtcgagt ttaggggctg
     6721 agttaaggag gttggctctc gggctgagga acttgtttat tgcagcttat aatggttaca
     6781 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     6841 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatctgatca ctgcttgagc
     6901 ctaggagatc cgaaccagat aagtgaaatc tagttccaaa ctattttgtc atttttaatt
     6961 ttcgtattag cttacgacgc tacacccagt tcccatctat tttgtcactc ttccctaaat
     7021 aatccttaaa aactccattt ccacccctcc cagttcccaa ctattttgtc cgcccacagc
     7081 ggggcatttt tcttcctgtt atgtttttaa tcaaacatcc tgccaactcc atgtgacaaa
     7141 ccgtcatctt cggctacttt
//
