LOCUS       sequence-495604-        4674 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  for pegRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4674)
  AUTHORS   Sun C, Li H, Liu Y, Li Y, Gao R, Shi X, Fei H, Liu J, Liang R, Gao C
  TITLE     Iterative recombinase technologies for efficient and precise genome 
            engineering across kilobase to megabase scales.
  JOURNAL   Cell. 2025 Aug 1:S0092-8674(25)00800-1. doi: 
            10.1016/j.cell.2025.07.011.
  PUBMED    40763736
REFERENCE   2  (bases 1 to 4674)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2025 
            Aug 1:S0092-8674(25)00800-1. doi: 10.1016/j.cell.2025.07.011."
COMMENT     Sequence Label: pGSPE-ccdb
FEATURES             Location/Qualifiers
     source          1..4674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1775..2080
                     /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"
     ncRNA           2088..2155
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      2186..2434
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     primer_bind     complement(2453..2469)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2453..2469)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2466..2488)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2477..2493
                     /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(2501..2507,2508..2525,2526..2531))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2546..2567
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2684..2701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2855..3443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2935..2954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3614..4405,4406..4474))
                     /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     4237..4256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4475..4579)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4647..4665
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt catggagtca aagattcaaa
      421 tagaggacct aacagaactc gccgtaaaga ctggcgaaca gttcatacag agtctcttac
      481 gactcaatga caagaagaaa atcttcgtca acatggtgga gcacgacaca cttgtctact
      541 ccaaaaatat caaagataca gtctcagaag accaaagggc aattgagact tttcaacaaa
      601 gggtaatatc cggaaacctc ctcggattcc attgcccagc tatctgtcac tttattgtga
      661 agatagtgga aaaggaaggt ggctcctaca aatgccatca ttgcgataaa ggaaaggcca
      721 tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca
      781 tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgattggc
      841 agacatactg tcccacaaat gaagatggaa tctgtaaaag aaaacgcgtg aaataatgcg
      901 tctgacaaag gttaggtcgg ctgcctttaa tcaataccaa agtggtccct accacgatgg
      961 aaaaactgtg cagtcggttt ggctttttct gacgaacaaa taagattcgt ggccgacagg
     1021 tgggggtcca ccatgtgaag gcatcttcag actccaataa tggagcaatg acgtaagggc
     1081 ttacgaaata agtaagggta gtttgggaaa tgtccactca cccgtcagtc tataaatact
     1141 tagcccctcc ctcattgtta agggagcaaa atctcagaga gatagtccta gagagagaaa
     1201 gagagcaagt agcctagaag tagtcaaggc ggcgaagtat tcaggcacgt ggccaggaag
     1261 aagaaaagcc aagacgacga aaacaggtaa gagctaagca tctagataag ttgaaaacaa
     1321 tcttcaaaag tcccacatcg cttagataag aaaacgaagc tgagtttata tacagctaga
     1381 gtcgaagtag tgattgaaca aagcaccagt ggtctagtgg tagaatagta ccctgccacg
     1441 gtacagaccc gggttcgatt cccggctggt gcattagtga gaccgctgat ttttgcggta
     1501 taagaatata tactgatatg tatacccgaa gtatgtcaaa aggaggtatg ctatgaagca
     1561 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
     1621 aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa
     1681 cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac
     1741 ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa
     1801 aagagagagc cgttatcgtc tgtttgtgga tgttcagagt gatattattg acacgcccgg
     1861 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
     1921 actttacccg gtggtgcata tcggggatga aagctggcgt atgatgacaa ccgatatggc
     1981 cagtgtgccg gtttccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
     2041 catcaaaaac gccattaacc tgatgttctg gggaatataa ggtctcaggc cggcatggtc
     2101 ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat gggacttttt
     2161 tttgatatct ccggggctaa ttgaatatga agatgaagat gaaatatttg gtgtgtcaaa
     2221 taaaaagctg gtgtgcttaa gtttgtgttt ttttcttggc ttgttgtgtt atgaatttgt
     2281 ggctttttct aatattaaat gaatgtaaga tctcattata atgaataaac aaatgtttct
     2341 ataatccatt gtgaatgttt tgttggatct cttctgcagc atataactac tgtatgtgct
     2401 atggtatgga ctatggaata tgattaaaga taagaagctt ggcgtaatca tggtcatagc
     2461 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2521 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2581 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2641 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2701 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2761 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2821 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2881 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2941 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3001 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3061 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3121 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3181 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3241 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3301 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3361 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3421 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3481 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3541 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3601 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3661 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3721 taccatctgg ccccagtgct gcaatgatac cgcgagaacc acgctcaccg gctccagatt
     3781 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3841 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3901 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3961 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4021 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4081 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4141 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4201 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4261 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4321 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4381 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4441 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4501 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4561 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4621 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
