LOCUS       sequence-430987-        7833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor to integrate a SNAP-Tag at the 
            endogenous LC3 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7833)
  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 7833)
  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: SNAP-LC3 HRD
FEATURES             Location/Qualifiers
     source          1..7833
                     /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             3091..3156
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3157..3190
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3197..3245
                     /note="synthetic polyadenylation signal"
     CDS             complement(3246..3845)
                     /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(3329..3349)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3826..3845
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3852..4168)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3866..4001
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    4175..4208
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4217..4759
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEV
                     PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV
                     VKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAV
                     KEWLLAHEGHRLGKPGLG"
     CDS             4772..4792
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     polyA_signal    5217..5351
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5254..5273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5308..5327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  5380..5545
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      5729..6183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5816..5835)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6025..6046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6209..6313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6314..6382,6383..7174)
                     /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(6532..6551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7345..7833,1..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 gccccgggac accccgcctg
     2641 tggctcagcc tcccccgccc cgcgctgcca tctcctcagg gccgggcagc aggctcccga
     2701 gcgcccacag acccggggtg cggcccagcc cacaaccgtc acctcagggg cctcaggcgc
     2761 ccagcggtgc tgggcggggc tggggcacga ccgggagcat gcgcagagcg cgcgtttcgc
     2821 ccatcgcgca cgcgcacaca cctgctccgc ccccacgctg cgtgccgctg ctgggttccg
     2881 ccacgcccgt catggcggcg gccccggccg gctctggccc cgcccctcgg tgacgcgtcg
     2941 cgagtcacct gaccaggctg cgggctgagg agatacaagg gaagtggcta tcgccagagt
     3001 cggattcgcc gccgcagcag ccgccgcccc cgggagccgc cgggaccctc gcgtcgtcgc
     3061 cgccgccgcc gcccagatcc ctgcaccatg gactacaaag accatgacgg tgattataaa
     3121 gatcatgata tcgattacaa ggatgacgat gacaagataa cttcgtataa tgtatgctat
     3181 acgaagttat aagcttcaca caaaaaacca acacacagat gtaatgaaaa taaagatatt
     3241 ttatttcagg caccgggctt gcgggtcatg caccaggtgc gcggtccttc gggcacctcg
     3301 acgtcggcgg tgacggtgaa gccgagccgc tcgtagaagg ggaggttgcg gggcgcggag
     3361 gtctccagga aggcgggcac cccggcgcgc tcggccgcct ccactccggg gagcacgacg
     3421 gcgctgccca gacccttgcc ctggtggtcg ggcgagacgc cgacggtggc caggaaccac
     3481 gcgggctcct tgggccggtg cggcgccagg aggccttcca tctgttgctg cgcggccagc
     3541 cgggaaccgc tcaactcggc catgcgcggg ccgatctcgg cgaacaccgc ccccgcttcg
     3601 acgctctccg gcgtggtcca gaccgccacc gcggcgccgt cgtccgcgac ccacaccttg
     3661 ccgatgtcga gcccgacgcg cgtgaggaag agttcttgca gctcggtgac ccgctcgatg
     3721 tggcggtccg gatcgacggt gtggcgcgtg gcggggtagt cggcgaacgc ggcggcgagg
     3781 gtgcgtacgg ccctggggac gtcgtcgcgg gtggcgaggc gcaccgtggg cttgtactcg
     3841 gtcatgagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat
     3901 agctcagagg ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg
     3961 ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg
     4021 actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     4081 ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct
     4141 gctggggagc ctggggactt tccacaccgg atccataact tcgtataatg tatgctatac
     4201 gaagttatcc ggtaccgaca aagactgcga aatgaagcgc accaccctgg atagccctct
     4261 gggcaagctg gaactgtctg ggtgcgaaca gggcctgcac cgtatcatct tcctgggcaa
     4321 aggaacatct gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc
     4381 agagccactg atgcaggcca ccgcctggct caacgcctac tttcaccagc ctgaggccat
     4441 cgaggagttc cctgtgccag ccctgcacca cccagtgttc cagcaggaga gctttacccg
     4501 ccaggtgctg tggaaactgc tgaaagtggt gaagttcgga gaggtcatca gctacagcca
     4561 cctggccgcc ctggccggca atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg
     4621 aaatcccgtg cccattctga tcccctgcca ccgggtggtg cagggcgacc tggacgtggg
     4681 gggctacgag ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg gccacagact
     4741 gggcaagcct gggctgggtg agccaaccac tgaggatctg tactttcaga gcgataacgc
     4801 gatcgcttcc ggtaccccgt cggagaagac cttcaagcag cgccgcacct tcggtgagtg
     4861 tcgccgcgag ggcggcgggt gcggcggggc cggggtccga gctgtggagg gcggcagggc
     4921 ctgggacgcc gtgaggggtc ggggccgagc cgggaggccg agcggggccg gtggctgccg
     4981 gccggcgggg ccgagggatg cggggcccgg ggccccgtga gggacccagg ccgggaccga
     5041 gccgggaggg ccaggggctg gtctgggccg gggcggcctg cggacctctc ggaggcctgg
     5101 gaggaggcag ccggcggagg gcgggcgggg gccggtgcgc tcggggcccc gggcctgctg
     5161 aatcaccccg cgccctccgc ggcggggtgc ctgccgtacc ccggccaccg ccgcccaact
     5221 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5281 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5341 atgtctggat ctgatcactg cttgagccta ggagatccga accagataag tgaaatctag
     5401 ttccaaacta ttttgtcatt tttaattttc gtattagctt acgacgctac acccagttcc
     5461 catctatttt gtcactcttc cctaaataat ccttaaaaac tccatttcca cccctcccag
     5521 ttcccaacta ttttgtccgc ccacagcggg gcatttttct tcctgttatg tttttaatca
     5581 aacatcctgc caactccatg tgacaaaccg tcatcttcgg ctactttttc tctgtcacag
     5641 aatgaaaatt tttctgtcat ctcttcgtta ttaatgtttg taattgactg aatatcaacg
     5701 cttatttgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     5761 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5821 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5881 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5941 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     6001 acgttggagt ccacgttctt aatagtggac tcttgttcca aactggaaca acactcaacc
     6061 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     6121 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     6181 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     6241 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     6301 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     6361 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     6421 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6481 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     6541 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6601 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6661 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6721 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6781 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6841 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6901 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6961 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     7021 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     7081 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     7141 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     7201 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     7261 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     7321 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     7381 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7441 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     7501 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7561 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7621 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7681 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7741 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7801 cggaacagga gagcgcacga gggagcttcc agg
//
