LOCUS       sequence-429514-        8181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homologous recombination donor to integrate a 3xFLAG-HaloTag at the 
            endogenous LC3B locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8181)
  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 8181)
  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-LC3B HRD
FEATURES             Location/Qualifiers
     source          1..8181
                     /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..5107
                     /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             5120..5140
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     polyA_signal    5565..5699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5602..5621)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5656..5675
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  5728..5893
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      6077..6531
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6164..6183)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6373..6394
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6557..6661
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6662..6730,6731..7522)
                     /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(6880..6899)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7693..8181,1..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 ggtaccatgg cagaaatcgg tactggcttt ccattcgacc cccattatgt
     4261 ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt
     4321 gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt
     4381 tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc
     4441 agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct
     4501 gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg
     4561 ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat
     4621 cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac
     4681 cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat
     4741 gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa
     4801 tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc
     4861 agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc
     4921 gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct
     4981 ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca
     5041 agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcga cgctcgagat
     5101 ttccggcgag ccaaccactg aggatctgta ctttcagagc gataacgcga tcgcttccgg
     5161 taccccgtcg gagaagacct tcaagcagcg ccgcaccttc ggtgagtgtc gccgcgaggg
     5221 cggcgggtgc ggcggggccg gggtccgagc tgtggagggc ggcagggcct gggacgccgt
     5281 gaggggtcgg ggccgagccg ggaggccgag cggggccggt ggctgccggc cggcggggcc
     5341 gagggatgcg gggcccgggg ccccgtgagg gacccaggcc gggaccgagc cgggagggcc
     5401 aggggctggt ctgggccggg gcggcctgcg gacctctcgg aggcctggga ggaggcagcc
     5461 ggcggagggc gggcgggggc cggtgcgctc ggggccccgg gcctgctgaa tcaccccgcg
     5521 ccctccgcgg cggggtgcct gccgtacccc ggccaccgcc gcccaacttg tttattgcag
     5581 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5641 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct
     5701 gatcactgct tgagcctagg agatccgaac cagataagtg aaatctagtt ccaaactatt
     5761 ttgtcatttt taattttcgt attagcttac gacgctacac ccagttccca tctattttgt
     5821 cactcttccc taaataatcc ttaaaaactc catttccacc cctcccagtt cccaactatt
     5881 ttgtccgccc acagcggggc atttttcttc ctgttatgtt tttaatcaaa catcctgcca
     5941 actccatgtg acaaaccgtc atcttcggct actttttctc tgtcacagaa tgaaaatttt
     6001 tctgtcatct cttcgttatt aatgtttgta attgactgaa tatcaacgct tatttgcagc
     6061 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6121 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6181 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6241 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6301 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6361 acgttcttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     6421 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     6481 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     6541 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6601 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6661 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6721 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6781 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6841 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6901 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6961 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     7021 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     7081 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     7141 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     7201 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     7261 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     7321 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     7381 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     7441 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7501 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7561 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7621 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7681 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7741 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7801 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     7861 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7921 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7981 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     8041 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     8101 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     8161 gcgcacgagg gagcttccag g
//
