LOCUS       sequence-218864-        8021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of the CDK4 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8021)
  TITLE     Salmon Lab plasmids
REFERENCE   2  (bases 1 to 8021)
  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: pCLX-PGK-hCDK4
FEATURES             Location/Qualifiers
     source          1..8021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..21
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        26..536
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     455..473
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    537..561
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1479..1503)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    1521..2109
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1574..1594)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1992..2003)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(2252..2285)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     LTR             2305..2485
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(2523..2542)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2588..2643
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2642..2661)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3004..3020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3004..3020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3017..3039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3028..3044
                     /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(3052..3058,3059..3076,3077..3082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3097..3118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3177..3372
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3223..3358
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3378..3512
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3415..3434)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3469..3488
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3580..3597)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3751..4339)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3831..3850)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4510..5301,5302..5370))
                     /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     5133..5152
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5371..5475)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5506..5885
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     LTR             5887..6144
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6191..6316
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6808..6925
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    7285..7518
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7703..7747
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 caactttgta tagaaaagtt gccacggggt tggggttgcg ccttttccaa ggcagccctg
       61 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
      121 ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
      181 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
      241 tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg
      301 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
      361 gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg
      421 gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg
      481 agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggcaa
      541 gtttgtacaa aaaagcaggc ttcaccatgg ctacctctcg atatgagcca gtggctgaaa
      601 ttggtgtcgg tgcctatggg acagtgtaca aggcccgtga tccccacagt ggccactttg
      661 tggccctcaa gagtgtgaga gtccccaatg gaggaggagg tggaggaggc cttcccatca
      721 gcacagttcg tgaggtggct ttactgaggc gactggaggc ttttgagcat cccaatgttg
      781 tccggctgat ggacgtctgt gccacatccc gaactgaccg ggagatcaag gtaaccctgg
      841 tgtttgagca tgtagaccag gacctaagga catatctgga caaggcaccc ccaccaggct
      901 tgccagccga aacgatcaag gatctgatgc gccagtttct aagaggccta gatttccttc
      961 atgccaattg catcgttcac cgagatctga agccagagaa cattctggtg acaagtggtg
     1021 gaacagtcaa gctggctgac tttggcctgg ccagaatcta cagctaccag atggcactta
     1081 cacccgtggt tgttacactc tggtaccgag ctcccgaagt tcttctgcag tccacatatg
     1141 caacacctgt ggacatgtgg agtgttggct gtatctttgc agagatgttt cgtcgaaagc
     1201 ctctcttctg tggaaactct gaagccgacc agttgggcaa aatctttgac ctgattgggc
     1261 tgcctccaga ggatgactgg cctcgagatg tatccctgcc ccgtggagcc tttcccccca
     1321 gagggccccg cccagtgcag tcggtggtac ctgagatgga ggagtcggga gcacagctgc
     1381 tgctggaaat gctgactttt aacccacaca agcgaatctc tgcctttcga gctctgcagc
     1441 actcttatct acataaggat gaaggtaatc cggagtgaac ccagctttct tgtacaaagt
     1501 ggtgataatc gaattccgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     1561 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     1621 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     1681 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     1741 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     1801 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     1861 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga
     1921 cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     1981 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     2041 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     2101 cctccccgca tcgggaattc ccgcggttcg ctttaagacc aatgacttac aaggcagctg
     2161 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     2221 gaagacaaga tgggatcaat tcaccatggg aataacttcg tatagcatac attatacgaa
     2281 gttatgctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     2341 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     2401 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     2461 tagtcagtgt ggaaaatctc tagcagcatc tagttgaatc gccagtgtgt cgagctagca
     2521 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     2581 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     2641 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     2701 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     2761 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     2821 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta
     2881 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     2941 atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc
     3001 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3061 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3121 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat
     3181 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3241 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3301 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3361 ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3421 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3481 aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc
     3541 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3601 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3661 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3721 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3781 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3841 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3901 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3961 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4021 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4081 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4141 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4201 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4261 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4321 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4381 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4441 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4501 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4561 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4621 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4681 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4741 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4801 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4861 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4921 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4981 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5041 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5101 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5161 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5221 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5281 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5341 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5401 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5461 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcgacat tgattattga
     5521 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     5581 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     5641 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     5701 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     5761 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     5821 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     5881 ccatgggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat cctgcatata
     5941 agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc
     6001 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     6061 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     6121 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
     6181 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
     6241 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
     6301 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
     6361 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
     6421 gctagaacga ttcgcagtta atactggcct gttagaaaca tcagaaggct gtagacaaat
     6481 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     6541 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     6601 tttagacaag atagaggaag agcaaaacaa aagtaagaaa aaagcacagc aagcagcagg
     6661 atcttcagac ctggaaattc cctacaatcc ccaaagtcaa ggagtagtag aatctatgaa
     6721 taaagaatta aagaaaatta taggacaggt aagagatcag gctgaacatc ttaagacagc
     6781 agtacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     6841 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     6901 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagaa atccactttg
     6961 gaaaggacca gcaaagctcc tctggaaagg tgaaggggca gtagtaatac aagataatag
     7021 tgacataaaa gtagtgccaa gaagaaaagc aaagatcatt agggattatg gaaaacagat
     7081 ggcaggtgat gattgtgtgg caagtagaca ggatgaggat tagaacatgg aaaagtttag
     7141 taaaacacca taaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     7201 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     7261 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     7321 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     7381 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     7441 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     7501 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     7561 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     7621 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     7681 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     7741 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     7801 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     7861 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     7921 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     7981 attagtgaac ggatctcgac ggtatctagt gattatcgga t
//
