LOCUS       sequence-349332-        4377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for endogenous tagging of human VPS35 at the C-terminus
            with halotag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4377)
  TITLE     iNDI donor vectors for endogenous tagging with Halotag
REFERENCE   2  (bases 1 to 4377)
  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"
COMMENT     Sequence Label: VPS35_Halo_C_allele
FEATURES             Location/Qualifiers
     source          1..4377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     183..205
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     197..214
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     198..214
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             666..1553
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     primer_bind     complement(1975..1991)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1975..1991)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1988..2010)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1999..2015
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2023..2029,2030..2047,2048..2053))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2068..2089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2206..2223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2377..2965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2457..2476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3136..3927,3928..3996))
                     /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     3759..3778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3997..4101)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4169..4187
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4225..4247)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4347..4366
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
       61 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
      121 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
      181 tacccagtca cgacgttgta aaacgacggc cagtgaattc tgagccgagc caattttgct
      241 tgttttaaag ggttgttttt tttttttttt ttttgatagt cagtaattgt tcaaactagg
      301 aattgtatcc ccatctttct tttttcataa ttactcaggt aattgatgag tgtaacagaa
      361 gctcctcaaa acagttttat taaattgcct ttcatttttt gtggtacgtg cttgatcatg
      421 aatttgtaca tattcttttg taggtaacaa ttcaggtttt aaaccagctt atccaaaaga
      481 ttcgagaaga cctcccgaat cttgaatcca gtgaagaaac agagcagatt aacaaacatt
      541 ttcataacac actggagcat ttgcgcttgc ggcgggaatc accagaatcc gaggggccaa
      601 tttatgaagg tctcatcctt ggtggaggag gttctggagg cggtggaagt ggtggcggag
      661 gtagcgccga gatcggcacc ggctttccct tcgatcctca ctacgtggaa gtgctgggcg
      721 agagaatgca ctatgtggac gtgggcccca gagatggaac ccctgtgctg tttctgcacg
      781 gcaaccctac cagcagctac gtgtggcgga acatcatccc tcacgtggcc cctacacaca
      841 gatgtatcgc ccctgacctg atcggcatgg gcaagagcga taagcccgac ctgggctact
      901 tcttcgacga ccacgtgcgg ttcatggacg cctttattga ggccctcggc ctggaagagg
      961 tggtgctggt tattcacgat tggggcagcg ccctgggatt ccactgggcc aagagaaacc
     1021 ccgagagagt gaagggaatc gccttcatgg aattcatcag gcccattcct acctgggacg
     1081 agtggcctga gttcgccaga gagacattcc aggccttcag aaccaccgac gtgggcagaa
     1141 agctgatcat cgaccagaac gtgttcatcg agggcaccct gcctatggga gtcgtcagac
     1201 cactgaccga ggtggaaatg gaccactaca gagagccctt tctgaacccc gtggaccggg
     1261 aacctctttg gagattcccc aacgagctgc ccattgccgg cgagcctgct aatattgtgg
     1321 ccctggtgga agagtacatg gactggctgc atcagagccc cgtgcctaag ctgctgtttt
     1381 ggggaacacc cggcgtgctg attcctcctg ctgaagctgc cagactggcc aagagcctgc
     1441 ctaattgcaa ggccgtggat atcggccctg gcctgaatct gctgcaagag gacaaccccg
     1501 atctgatcgg ctccgagatt gccagatggc tgagcaccct ggaaatcagc ggataaaaag
     1561 gaaatagctc accatactcc tttccatgta catccagtga gggttttatt acgctaggtt
     1621 tcccttccat agattgtgcc tttcagaaat gctgaggtag gtttcccatt tcttacctgt
     1681 gatgtgtttt acccagcacc tccggacact caccttcagg accttaataa aattattcac
     1741 ttggtaagtg ttcaagtctt tctgatcacc ccaagtagca tgactgatct gcaatttaaa
     1801 attcctgtga tctgtaaaaa aaaaaaaaaa aaaaaacaca aaacccacaa gcacttatct
     1861 tggctactaa tgaagctctc cttttttttg tttgtttgtt tgcttcattg ttgattgtgt
     1921 attttcttca ttcctgggga gtactaaccc aaaagcaagc ttggcgtaat catggtcata
     1981 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2041 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2101 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2161 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2221 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2281 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2341 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2401 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2461 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2521 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2581 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2641 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2701 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2761 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2821 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2881 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2941 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3001 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3061 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3121 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3181 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3241 cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga
     3301 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3361 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3421 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3481 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3541 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3601 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3661 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3721 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3781 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3841 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3901 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3961 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4021 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4081 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4141 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     4201 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4261 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     4321 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgca
//
