LOCUS       sequence-435058-        7010 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 7010)
  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 7010)
  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: pPB-W-ccdB-Z
FEATURES             Location/Qualifiers
     source          1..7010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1680..1714
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             complement(2021..2326)
                     /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(2123..2142)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2668..3327)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3242..3261
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(3381..3387,3388..3405,3406..3411))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     repeat_region   3641..3703
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     4726..4742
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4726..4742
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4776..4792)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4777..4793)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(4822..4842)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4822..4840)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4861..4877)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4861..4877)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4874..4896)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4885..4901
                     /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(4909..4915,4916..4933,4934..4939))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4954..4975
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5092..5109)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5263..5851)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5343..5362)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6022..6813,6814..6882))
                     /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     6645..6664
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6883..6987)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcgt
      661 ttaaacggcg cgcctctgca cgacaggtct gccagcttac atttacccaa actgtccatt
      721 actggaacct atgatctgaa gagcgtcctg ggtcaactgg gcatcactaa ggtcttcagc
      781 aatggggctg acctctccgg ggtcacagag gaggcacccc tgaagctctc caaggtgaga
      841 tcaccctgac gaccttgttg caccctggta tctgtaggga agaatgtgtg ggggctgcag
      901 ctctgtcctg aggctgagga aggggccgag ggaaacaaat gaagacccag gctgagctcc
      961 tgaagatgcc cgtgattcac tgacacggga cgtggtcaaa cagcaaagcc aggcagggga
     1021 ctgctgtgca gctggcactt tcggggcctc ccttgaggtt gtgtcactga ccctgaattt
     1081 caactttgcc caagaccttc tagacattgg gccttgattt atccatactg acacagaaag
     1141 gtttgggcta agttgtttca aaggaatttc tgactccttc gatctgtgag atttggtgtc
     1201 tgaattaatg aatgatttca gctaaagatg acacttattt tggaaaacta aaggcgacca
     1261 atgaacaact gcagttccat gaatggctgc attatcttgg ggtctgggca ctgtgaaggt
     1321 cactgccagg gtccgtgtcc tcaaggagct tcaagccgtg tactagaaag gagagagccc
     1381 tggaggcaga cgtggagtga cgatgctctt ccctgttctg agttgtgggt gcacctgagc
     1441 agggggagag gcgcttgtca ggaagatgga cagaggggag ccagccccat cagccaaagc
     1501 cttgaggagg agcaaggcct atgtgacagg gagggagagg atgtgcaggg ccagggccgt
     1561 ccagggggag tgagcgcttc ctgggaggtg tccacgtgag ccttgctcga ggcctgggat
     1621 cagccttaca acgtgtctct gcttctctcc cctccaggcc gtgcataagg ctgtgttaac
     1681 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
     1741 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
     1801 cgtgaggcgt gcttgtcaat gcggtaagtg tcactgattt tgaactataa cgaccgcgtg
     1861 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
     1921 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
     1981 tagatatcaa ctagaatgct agctgcctga gacggtcgac ttatattccc cagaacatca
     2041 ggttaatggc gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga
     2101 taacggagac tggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga
     2161 tatgcaccac cgggtaaagt tcacgggaga ctttatctga cagcagacgt gcactggcca
     2221 gggggatcac catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca
     2281 gacgataacg gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagtccctg
     2341 ttctcgtcag caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc
     2401 tgattttccg ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg
     2461 cttaccagac cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt
     2521 caactgtcac tgtaatacgc tgcttcatag cacacctctt tttgacatac ttcgggtata
     2581 catatcagta tatattctta taccgcaaaa atcagcgcgc aaatacgcat actgttatct
     2641 ggcttttagt aagccggatc cacgcgttta cgccccgccc tgccactcat cgcagtactg
     2701 ttgtaattca ttaagcattc tgccgacatg gaagccatca cagacggcat gatgaacctg
     2761 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     2821 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     2881 gggattggct gacacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     2941 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     3001 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     3061 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg
     3121 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     3181 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     3241 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     3301 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgccgg
     3361 atcctaactc aaaatccaca cattatacga gccggaagca taaagtgtaa agcctggggt
     3421 gcctaatgcg gccgccgtct cacagatgta caaagtggta ctagttaaaa gttttgttac
     3481 tttatagaag aaattttgag tttttgtttt tttttaataa ataaataaac ataaataaat
     3541 tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta atatctattc
     3601 aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa ttttacgcat
     3661 gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaacca tcgacgagaa
     3721 agggactgaa gctgctgggg ccatgttttt agaggccata cccatgtcta tcccccccga
     3781 ggtcaagttc aacaaaccct ttgtcttctt aatgattgaa caaaatacca agtctcccct
     3841 cttcatggga aaagtggtga atcccaccca aaaataactg cctctcgctc ctcaacccct
     3901 cccctccatc cctggccccc tccctggatg acattaaaga agggttgagc tggtccctgc
     3961 ctgcatgtga ctgtaaatcc ctcccatgtt ttctctgagt ctccctttgc ctgctgaggc
     4021 tgtatgtggg ctccaggtaa cagtgctgtc ttcgggcccc ctgaactgtg ttcatggagc
     4081 atctggctgg gtaggcacat gctgggcttg aatccagggg ggactgaatc ctcagcttac
     4141 ggacctgggc ccatctgttt ctggagggct ccagtcttcc ttgtcctgtc tttgagtccc
     4201 caagaaggaa tcacagggga ggaaccagat accagccatg accccaggct ccaccaagca
     4261 tcttcatgtc cccctgctca tcccccactc ccccccaccc agagttgctc atcctgccag
     4321 ggctggctgt gcccacccca aggctgccct cctggggacc ccagaactgc ctgatcgtgc
     4381 cgtggcccag ttttgtggca tctgcagcaa cacaagagag aggacaatgt cctcctcttg
     4441 acccgctgtc acctaaccag actcgggccc tgcacctctc aggcacttct ggaaaatgac
     4501 tgaggcagat tcttcctgaa gcccattctc catggggcaa caaggacacc tattctgtcc
     4561 ttgtccttcc atcgctgccc cagaaagcct cacatatctc cgtttagaat caggtccctt
     4621 ctccccagat gaagaggagg gtctctgctt tgttttctct atctcctcct cagacttgac
     4681 caggcccagc aggccccaga agaccattaa ttaaggccgg gccgctctag aactagtgga
     4741 tcccccgggc tgcaggaatt cgatatcaag cttatcgata ccgtcgacct cgaggggggg
     4801 cccggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
     4861 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4921 cgggagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4981 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5041 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5101 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5161 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5221 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5281 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5341 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5401 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5461 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5521 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5581 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5641 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5701 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5761 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5821 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5881 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5941 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6001 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6061 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6121 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6181 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6241 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6301 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6361 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6421 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6481 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6541 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6601 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6661 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6721 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6781 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6841 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6901 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6961 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
