LOCUS       sequence-489191-        6612 bp    DNA     circular SYN 14-OCT-2025
DEFINITION  Express HiBit-VPS4A in Mammalian Cells under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6612)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6612)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: HiBit-VPS4A Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     288..307
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    482..530
                     /note="synthetic polyadenylation signal"
     promoter        686..790
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(791..859,860..1651)
                     /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(1009..1028)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1809..2397)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1889..1908)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    2513..2796
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     enhancer        2987..3366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3367..3570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3521..3541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3706..3838
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3821..3840
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     3882..3901
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3882..3900
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5407..5425)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    5462..5583
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5473..5492)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5527..5546
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        5828..6185
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5828..6019
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5851..5871)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6036..6171
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(6236..6612,1..418)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6290..6309)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
       61 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
      121 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
      181 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
      241 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
      301 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
      361 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
      421 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgatggccg
      481 caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagc
      541 gataaggatc ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt
      601 catccggggt cagcaccgtt tctgcggact ggctttctac gtaatggttt cttagacgtc
      661 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
      721 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
      781 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      841 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      901 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      961 ttttcgcccc gaagaacgtt ttccaatgat gagcactttc aaagttctgc tatgtggcgc
     1021 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     1081 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     1141 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     1201 gacaactatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     1261 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     1321 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     1381 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     1441 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     1501 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     1561 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     1621 gataggtgcc tcactgatta agcattggta attcgaaatg accgaccaag cgacgcccaa
     1681 ccggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1741 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1801 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1861 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1921 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1981 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2041 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2101 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2161 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2221 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     2281 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2341 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa
     2401 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2461 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaaat
     2521 acaggaacgc acgctggatg gcccttcgct gggatggtga aaccatgaaa aatggcagct
     2581 tcagtggatt aagtgggggt aatgtggcct gtaccctctg gttgcatagg tattcatacg
     2641 gttaaaattt atcaggcgcg attgcggcag tttttcgggt ggtttgttgc catttttacc
     2701 tgtctgctgc cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta agggattatg
     2761 gtaaatccac ttactgtctg ccctcgtagc catcgagata aaccgcagta ctccggccac
     2821 gatgcgtccg gcgtagagga tcgagatctt caatattggc cattagccat attattcatt
     2881 ggttatatag cataaatcaa tattggctat tggccattgc atacgttgta tctatatcat
     2941 aatatgtaca tttatattgg ctcatgtcca atatgaccgc catgttggca ttgattattg
     3001 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3061 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3241 ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3301 tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3361 accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg
     3421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     3481 cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt
     3541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat cactagaagc
     3601 tttattgcgg tagtttatca cagttaaatt gctaacgcag tcagtgcttc tgacacaaca
     3661 gtctcgaact taagctgcag aagttggtcg tgaggcactg ggcaggtaag tatcaaggtt
     3721 acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag aagactcttg
     3781 cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt
     3841 gtccactccc agttcaatta cagctcttaa ggctagagta ttaatacgac tcactatagg
     3901 gctagcgctc accatggtga gcggctggcg gctgttcaag aagattagcg ggagctccgg
     3961 tggctcgagc ggtgcgatcg ccatgacaac gtcaaccctc cagaaagcca ttgatctggt
     4021 gacgaaagcc acagaggagg acaaagccaa gaactacgag gaggcgctgc ggctgtacca
     4081 gcatgcggtg gagtacttcc tccacgctat caagtatgag gcccacagcg acaaggccaa
     4141 ggagagcatt cgagccaagt gcgtgcagta cctagaccgg gccgagaagc tgaaggatta
     4201 tttacgaagc aaagagaaac acggcaagaa gccagtcaaa gagaaccaga gtgagggcaa
     4261 gggcagtgac agtgacagtg aaggggataa tccggagaaa aagaaactgc aagaacagct
     4321 gatgggtgcc gtcgtgatgg agaagcccaa catacggtgg aacgacgtgg ccgggctgga
     4381 gggggccaag gaggccctca aagaagctgt cattttgcca atcaaattcc cacacttgtt
     4441 cacaggcaag cgcaccccct ggcgggggat tctgctgttc ggaccccctg gcacagggaa
     4501 atcctacctg gccaaagccg tggcaacaga ggccaacaac tccaccttct tctctgtgtc
     4561 ctcctcagat ctgatgtcca agtggctggg ggagagtgaa aagctggtca agaacctgtt
     4621 tgagctggcc aggcagcaca agccctccat catcttcatc gatgaggtgg attccctctg
     4681 cgggtcccga aatgaaaatg agagtgaggc cgcccggagg atcaaaacgg agttcttggt
     4741 ccagatgcag ggggtgggga ataacaatga tgggactctg gttcttggag ccacaaacat
     4801 cccatgggtg ttggattcgg ccatcaggag gaggtttgaa aaacgaattt atatcccctt
     4861 gccggaggaa gctgcccgcg cccagatgtt ccggttgcat ctcgggagca ctccccacaa
     4921 cctcacggat gcaaacatcc acgagctggc ccggaagacg gaaggctact cgggcgcgga
     4981 catcagcatc atcgtgcggg actctctcat gcagcccgtg aggaaggtgc agtcggccac
     5041 acacttcaaa aaggtctgtg gcccctctcg caccaacccc agcatgatga ttgatgacct
     5101 cctgactcca tgctcaccag gggacccagg agccatggag atgacttgga tggatgtccc
     5161 tggggacaaa ctcttagagc ctgtggtttg catgtcggac atgctgcggt ctctggccac
     5221 cacccggccc acggtgaatg cagacgacct cctgaaagtg aagaaattct cagaggactt
     5281 tgggcaagag agtgtttaaa cgaattcggg ctcggtaccc ggggatcctc tagagtcgac
     5341 ctgcaggcat gcaagctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     5401 ctgccaccgc tgagcaataa ctagcataac cccttggggc ggccgcttcg agcagacatg
     5461 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     5521 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     5581 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat gtgggaggtt
     5641 tttttaagca agtaaaacct ctacaaatgt ggtaaaatcg aattttaaca aaatattaac
     5701 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5761 atacgcggat ctgcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag
     5821 gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     5881 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5941 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     6001 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     6061 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     6121 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     6181 gcaaaaagct tgattcttct gacacaacag tctcgaactt aaggctagag ccaccatgat
     6241 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     6301 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     6361 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     6421 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     6481 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     6541 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     6601 gctgcatacg ct
//
