LOCUS       sequence-490270-        6190 bp    DNA     circular SYN 22-OCT-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6190)
  AUTHORS   Iliopoulos O, Kibel A, Gray S, Kaelin WG
  TITLE     Tumour suppression by the human von Hippel-Lindau gene product.
  JOURNAL   Nat Med. 1995 Aug . 1(8):822-6.
  PUBMED    7585187
REFERENCE   2  (bases 1 to 6190)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Med. 
            1995 Aug . 1(8):822-6."
COMMENT     Sequence Label: HA-VHL-pRc/CMV
FEATURES             Location/Qualifiers
     source          1..6190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        147..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        527..730
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     681..701
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     775..794
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        775..793
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             816..842
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             816..842
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             822..848
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     promoter        complement(1561..1579)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1562..1579)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1599..1616)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1605..1829
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1875..2388
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1962..1981)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2172..2193
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2411..2431)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2416..2745
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2596..2731
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2812..3606
                     /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(2866..2885)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3476..3495
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3780..3914
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3817..3836)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3871..3890
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3945..3961)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3945..3961)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3958..3980)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3969..3985
                     /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(3993..3999,4000..4017,4018..4023))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4038..4059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4176..4193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4347..4935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4427..4446)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5106..5897,5898..5966))
                     /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     5729..5748
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5967..6071)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6146..6165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
       61 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
      121 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      541 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      601 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      661 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      721 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      781 gactcactat agggagaccc aagcttacca tggcctaccc ctacgacgtg cccgactacg
      841 cctccctcgg atccgccacc atgccccgga gggcggagaa ctgggacgag gccgaggtag
      901 gcgcggagga ggcaggcgtc gaagagtacg gccctgaaga agacggcggg gaggagtcgg
      961 gcgccgagga gtccggcccg gaagagtccg gcccggagga actgggcgcc gaggaggaga
     1021 tggaggccgg gcggccgcgg cccgtgctgc gctcggtgaa ctcgcgcgag ccctcccagg
     1081 tcatcttctg caatcgcagt ccgcgcgtcg tgctgcccgt atggctcaac ttcgacggcg
     1141 agccgcagcc ctacccaacg ctgccgcctg gcacgggccg ccgcatccac agctaccgag
     1201 gtcacctttg gctcttcaga gatgcaggga cacacgatgg gcttctggtt aaccaaactg
     1261 aattatttgt gccatctctc aatgttgacg gacagcctat ttttgccaat atcacactgc
     1321 cagtgtatac tctgaaagag cgatgcctcc aggttgtccg gagcctagtc aagcctgaga
     1381 attacaggag actggacatc gtcaggtcgc tctacgaaga tctggaagac cacccaaatg
     1441 tgcagaaaga cctggagcgg ctgacacagg agcgcattgc acatcaacgg atgggagatt
     1501 gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag agggccctat
     1561 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     1621 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     1681 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     1741 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     1801 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     1861 gaggggggat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1921 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1981 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2041 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2101 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2161 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2221 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2281 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aaatatttgc
     2341 ttatacaatc ttcctgtttt tggggctttt ctgattatca accggggtgg gtaccgagct
     2401 cgaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2461 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2521 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2581 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2641 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     2701 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     2761 tggatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     2821 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2881 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2941 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3001 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3061 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3121 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3181 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3241 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3301 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3361 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3421 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3481 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3541 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3601 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3661 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     3721 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     3781 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     3841 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3901 atcatgtctg gatccgtcga cctcgagagc ttggcgtaat catggtcata gctgtttcct
     3961 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4021 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4081 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4141 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5161 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5401 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5701 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     6121 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     6181 atctgctccc
//
