LOCUS       sequence-429537-        7153 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Homologous recombination donor to integrate a 3xFLAG-HaloTag at the 
            endogenous WIPI2 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7153)
  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 7153)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-WIPI2 HRD
FEATURES             Location/Qualifiers
     source          1..7153
                     /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             3081..3146
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    3147..3180
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3189..4079
                     /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             4092..4112
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     polyA_signal    4537..4671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4574..4593)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4628..4647
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  4700..4865
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      5049..5503
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5136..5155)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5345..5366
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5529..5633
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5634..5702,5703..6494)
                     /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(5852..5871)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6665..7153,1..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 ctcccaaaat gctgggatta
     2641 caggccaccg tgcctggcca ttgagtgtta ttaagaggag ggtacgcaga aaacagactc
     2701 ccgccagctc tgccccatgc gcatgagcgg ccgccgctcg ccctcagcca ggcccagcgc
     2761 gccccgacgc gtccccacgc ctgcgtgatc caccggaacc caagcccagg caggcggtga
     2821 gagggcgggg cctccgcagt gcgactgcgc cgctcgaggc cgtgcgtcgc gggggcgggg
     2881 ccgcgcaggc gtaccgggtg ccccggctct ggagcataaa caagagcggg gacgggatga
     2941 ggcggcggtt gatcccaggg tggcgagtgg cggcgaccga ggcggcgagc ggggcccggc
     3001 gccgaccctg agtgcagcct gacccgccct cgcgcgcgcg ccctccccgg ccgggcccac
     3061 tcgccgcgcg cccagccatg gactacaaag accatgacgg tgattataaa gatcatgata
     3121 tcgattacaa ggatgacgat gacaagataa cttcgtataa tgtatgctat acgaagttat
     3181 ccggtaccat ggcagaaatc ggtactggct ttccattcga cccccattat gtggaagtcc
     3241 tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc
     3301 tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga
     3361 cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg
     3421 gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg
     3481 aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc
     3541 gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct
     3601 gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg
     3661 gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg
     3721 tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg
     3781 accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca
     3841 tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc
     3901 tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa
     3961 gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca
     4021 acccggacct gatcggcagc gagatcgcgc gctggctgtc gacgctcgag atttccggcg
     4081 agccaaccac tgaggatctg tactttcaga gcgataacgc gatcgcttcc ggtaccaacc
     4141 tggcgagcca gagcggggag gccggcgccg gccagctgct cttcgccaac ttcaaccagg
     4201 acaacacgta aggccggggt cgggggtcgg agtcggggtg aggccagggt cggacccggg
     4261 ctagggggag ggcctcgctg ccaagctcgg cggcgtcgca ggctcggcct ccccgcgggc
     4321 caaggcgcgc agaggcgtcc ccaggggcgg gcccggggcg tgcagggagg ggcgccctcc
     4381 agggagaccc tcgggtgctg gcctggagct gcggtcccgg agcgggagag gtggtggaga
     4441 gggggctgct ttcactctct tgcggggagg ccccctggct tcagacttaa ctacttctcg
     4501 caaagttggg gccgaacttt ggggcttgac ttgtctaact tgtttattgc agcttataat
     4561 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4621 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ctgatcactg
     4681 cttgagccta ggagatccga accagataag tgaaatctag ttccaaacta ttttgtcatt
     4741 tttaattttc gtattagctt acgacgctac acccagttcc catctatttt gtcactcttc
     4801 cctaaataat ccttaaaaac tccatttcca cccctcccag ttcccaacta ttttgtccgc
     4861 ccacagcggg gcatttttct tcctgttatg tttttaatca aacatcctgc caactccatg
     4921 tgacaaaccg tcatcttcgg ctactttttc tctgtcacag aatgaaaatt tttctgtcat
     4981 ctcttcgtta ttaatgtttg taattgactg aatatcaacg cttatttgca gcctgaatgg
     5041 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5101 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5161 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5221 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5281 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5341 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5401 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5461 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     5521 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5581 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5641 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5701 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5761 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5821 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5881 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5941 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6001 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6061 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6121 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6181 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6241 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6301 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6361 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6421 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6481 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6541 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6601 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6661 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6721 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6781 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6841 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6901 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6961 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7021 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7081 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7141 gggagcttcc agg
//
