LOCUS       sequence-434964-        7062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  golden gate cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7062)
  AUTHORS   Delhaye L, Moschonas GD, Fijalkowska D, Verhee A, De Sutter D, Van 
            de Steene T, De Meyer M, Grzesik H, Van Moortel L, De Bosscher K, 
            Jacobs T, Eyckerman S
  TITLE     Leveraging a self-cleaving peptide for tailored control in proximity
            labeling proteomics.
  JOURNAL   Cell Rep Methods. 2024 Jul 15;4(7):100818. doi: 
            10.1016/j.crmeth.2024.100818. Epub 2024 Jul 9.
  PUBMED    38986614
REFERENCE   2  (bases 1 to 7062)
  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"; journalName: "Cell Rep 
            Methods."; date: "2024-07-15"; volume: "4(7):100818. doi"; pages: " 
            10.1016/j.crmeth.2024.100818. Epub 2024 Jul 9"
COMMENT     Sequence Label: pLV-W-ccdB-Z
FEATURES             Location/Qualifiers
     source          1..7062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..29
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13..29
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     48..68
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        50..68
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        96..322
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             323..503
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    550..675
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1168..1401
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1586..1630
                     /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"
     misc_feature    1896..2013
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2031..2051
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             complement(2179..2484)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2281..2300)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2826..3485)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3400..3419
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3539..3545,3546..3563,3564..3569))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     LTR             3676..3909
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3981..4102
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4018..4037)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4072..4091
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4142..4277
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4297..4316)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4298..4316)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4326..4343)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4326..4342)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4335..4357)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4484..4939
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4571..4590)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4781..4802
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4965..5069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5070..5138,5139..5930)
                     /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(5288..5307)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6101..6689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6590..6609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6843..6860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6977..6998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7013..7018,7019..7036,7037..7043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(7051..7062,1..5)
                     /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)."
     primer_bind     join(7056..7062,1..16)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
       61 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta
      121 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
      181 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
      241 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
      301 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct
      361 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
      421 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
      481 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
      541 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
      601 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
      661 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
      721 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
      781 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
      841 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
      901 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
      961 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct
     1021 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     1081 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     1141 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     1201 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     1261 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     1321 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     1381 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     1441 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     1501 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     1561 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     1621 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     1681 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     1741 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     1801 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat
     1861 tcgattagtg aacggatctc gacggtatcg ctagctttta aaagaaaagg ggggattggg
     1921 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     1981 ttacaaaaac aaattacaaa aattcaaaat tttactagtg attatcggat caactttgta
     2041 tagaaaagtt gggtaccgtc gacccaagct tcgcggatcc gcccgtaagc actaatacta
     2101 gtagtactgt cacttcctcc cgggagggtg cagagttcga agcatgcact caccactgca
     2161 gtgcctgaga cggtcgactt atattcccca gaacatcagg ttaatggcgt ttttgatgtc
     2221 attttcgcgg tggctgagat cagccacttc ttccccgata acggagactg gcacactggc
     2281 catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc
     2341 acgggagact ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc
     2401 gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt
     2461 ataggtgtaa accttaaact gcatttcacc agtccctgtt ctcgtcagca aaagagccgt
     2521 tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct ttccagcgtt
     2581 cggcacgcag acgacgggct tcattctgca tggttgtgct taccagaccg gagatattga
     2641 catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg taatacgctg
     2701 cttcatagca cacctctttt tgacatactt cgggtataca tatcagtata tattcttata
     2761 ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa gccggatcca
     2821 cgcgtttacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
     2881 ccgacatgga agccatcaca gacggcatga tgaacctgaa tcgccagcgg catcagcacc
     2941 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
     3001 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga cacgaaaaac
     3061 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
     3121 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
     3181 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
     3241 agctcaccgt ctttcattgc catacggaat tccggatgag cattcatcag gcgggcaaga
     3301 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
     3361 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
     3421 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
     3481 tccattttag cttccttagc tcctgaaaat ctcgccggat cctaactcaa aatccacaca
     3541 ttatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgcggc cgccgtctca
     3601 cagaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     3661 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     3721 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     3781 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     3841 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     3901 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     3961 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     4021 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4081 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     4141 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4201 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4261 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     4321 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4381 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4441 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     4501 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4561 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4621 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4681 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4741 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4801 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4861 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4921 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4981 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5041 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5101 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5161 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5221 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5281 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5341 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5401 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5461 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5521 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5581 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5641 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5701 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5761 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5821 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5881 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5941 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6001 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6061 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6121 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6181 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6241 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6301 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6361 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6421 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6481 tacagcgtga gctatgagaa agcgccacgc ttcccgaaga gagaaaggcg gacaggtatc
     6541 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6601 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6661 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6721 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6781 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6841 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     6901 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     6961 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     7021 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg at
//
