LOCUS       sequence-364201-        5642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chromosomal transfer helper plasmid with sgRNAs (one fixed, one 
            flexible) for Vibrio natriegens genome editing protocol..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5642)
  AUTHORS   Teufel M, Klein CA, Mager M, Sobetzko P
  TITLE     A multifunctional system for genome editing and large-scale 
            interspecies gene transfer.
  JOURNAL   Nat Commun. 2022 Jun 14;13(1):3430. doi: 10.1038/s41467-022-30843-1.
  PUBMED    35701417
REFERENCE   2  (bases 1 to 5642)
  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: "Nat 
            Commun."; date: "2022-06-14"; volume: "13(1):3430. doi"; pages: " 
            10.1038/s41467-022-30843-1"
COMMENT     Sequence Label: pDropVn
FEATURES             Location/Qualifiers
     source          1..5642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    20..38
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    147..168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(183..188,189..206,207..213)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    221..237
                     /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     226..248
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     245..261
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     245..261
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    274..330
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(331..348)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(331..347)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(340..362)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(556..575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             922..1227
                     /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     1106..1125
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1335..1410
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2862..2933
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2949..2976
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2987..3520)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(3539..3586)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     protein_bind    3690..3708
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3715..3733
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        3764..3839
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3859..3929
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      4381..4603
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4651..5601
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFKIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 tcgctgtagc acctgaagtt ccctatcagt gatagagatt gacatcccta tcagtgatag
       61 agatactgag cactgagacc cagtggtatg ggcagctggc acgacaggtt tcccgactgg
      121 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      181 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      241 cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca ggtcgactct
      301 agaggatccc cgggtaccga gctcgaattc actggccgtc gttttacaac gtcgtgactg
      361 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagga catccccctt tcgccagctg
      421 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
      481 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
      541 atggtgcact ctcagtacaa tctgctctga tgccgcatag tcgcgtggat ccggcttact
      601 aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac
      661 tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg
      721 acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc
      781 tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg
      841 aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg
      901 acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt
      961 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg
     1021 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
     1081 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgcctgtc
     1141 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
     1201 attaacctga tgttctgggg aatataatta agccagcccc gacacccgcc aacacccgct
     1261 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgacccac
     1321 tcaagtgggt ctcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     1381 caacttgaaa aagtggcacc gagtcggtgc tttttttacg gtcatgtaaa aaaaaacccc
     1441 gcccctgaca gggcggggtt ttttttcagt caagcctctc ttgacatcgc tagaaattgt
     1501 cttaagatat gttgcttatt taatgaaacg gcaggttctc ttgacatcgc tagaaattgt
     1561 cttaagatat gttgcttatt taatgaaasg scarswtsgk mtcwtakcwr skaswkdrwb
     1621 mgsasmwwym ctagrkgcgt aaaggcgaag cagtgatcaa agaatttatg cgcttcaaag
     1681 ttcacatgga aggttctatg aacggccacg aatttgaaat tgaaggtgaa ggcgaaggtc
     1741 gtccatacga aggtactcaa accgcaaaac tgaaagttac caaaggtggt ccactgccat
     1801 tttcttggga tattctgtct ccacaattta tgtacggttc tcgtgcattt atcaaacacc
     1861 cagcagatat tccagactac tacaaacaat cttttccgga aggtttcaaa tgggaacgtg
     1921 ttatgaattt tgaagatggt ggtgcagtta cggttaccca agatacctct ctggaagatg
     1981 gtactctgat ctacaaagtt aaactgcgtg gtactaactt tccaccagat ggtccagtta
     2041 tgcagaaaaa aaccatgggt tgggaagcat ctaccgaacg tctgtaccca gaagatggcg
     2101 ttctgaaagg tgatatcaaa atggcactgc gtctgatatg cccgtagcta ctagcgggtt
     2161 tctaaaggtg aagcagtgat caaagaattt atgcgcttca aagttcacat ggaaggttct
     2221 atgaacggcc acgaatttga aattgaaggt gaaggcgaag gtcgtccata cgaaggtact
     2281 caaaccgcaa aactgaaagt taccaaaggt ggtccactgc cattttcttg ggatattctg
     2341 tctccacaat ttatgtacgg ttctcgtgca tttatcaaac acccagcaga tattccagac
     2401 tactacaaac aatcttttcc ggaaggtttc aaatgggaac gtgttatgaa ttttgaagat
     2461 ggtggtgcag ttacggttac ccaagatacc tctctggaag atggtactct gatctacaaa
     2521 gttaaactgc gtggtactaa ctttccacca gatggtccag ttatgcagaa aaaaaccatg
     2581 ggttgggaag catctaccga acgtctgtac ccagaagatg gcgttctgaa aggtgatatc
     2641 aaaatggcac tgcgtctgaa agatggcggt cgttacctgg cagatttcaa aaccacctac
     2701 aaagcgaaaa aaccagttca aatgccaggt gcatacaacg ttgatcgtaa actggatatt
     2761 accagccaca acgaagatta caccgttgtt gaacaatacg aacgttctga aggccgtcac
     2821 tctaccggtg gtatggatga actgtacaaa taaccaggca tcaaataaaa cgaaaggctc
     2881 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga
     2941 gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta taatgattag gtggcggtgc
     3001 ttgggtcgat atcaaagtgc atcacttctt cccgtatgcc caactttgta tagagagcca
     3061 ctgcgggatc gtcaccgtaa tctgcttgca cgtagatcac ataagcacca agcgcgttgg
     3121 cctcatgctt gaggagattg atgagcgcgg tggcaatgcc ctgcctccgg tgctctccgg
     3181 agactgcgag atcatagata tagatctcac tacgcggctg ctcaaacttg ggcagaacgt
     3241 aagccgcgag agcgccaaca accgcttctt ggtcgaaggc agcaagcgca atgaatgtct
     3301 tactacggag caagttcccg aggtaatcgg agtccggctg atgttgggag taggtggcta
     3361 catctccgaa ctcacgaccg aaaagatcaa gagcagcccg catggatttg acttggtcag
     3421 ggccgagcct acatgtgcga atgatgccca tacttgagcc acctaacttt gttttagggc
     3481 gactgccctg ctgcgtaaca tcgttgctgc tgcgtaacat ggtttagttc ctcaccttgt
     3541 cgtattatac tatgccgata tactatgccg atgattaatt gtcaacacaa ctccggcagg
     3601 acttgttcag aacgctcggt cttgcacacc gggcgttttt tctttgtgag tccaggcaag
     3661 ctttcgctaa ggatgatttc tggaattctt ccctatcagt gatagagatt gacatcccta
     3721 tcagtgatag agatactgag cacgctcgat cgctaatcgt tatgttttag agctagaaat
     3781 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     3841 ttttttcaag ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     3901 tttatctgtt gtttgtcggt gaacgctctg ggtgcattag ccagtctgaa tgacctgtca
     3961 cgggataatc cgaagtggtc agactggaaa atcagagggc aggaactgct gaacagcaaa
     4021 aagtcagata gcaccacata gcagacccgc cataaaacgc cctgagaagc ccgtgacggg
     4081 cttttcttgt attatgggta gtttccttgc atgaatccat aaaaggcgcc tgtagtgcca
     4141 tttaccccca ttcactgcca gagccgtgag cgcagcgaac tgaatgtcac gaaaaagaca
     4201 gcgactcagg tgcctgatgg tcggagacaa aaggaatatt cagcgatttg cccgattgcg
     4261 gccgcaaccg agcttgcgag ggtgctactt aagcctttag ggttttaagg tctgttttgt
     4321 agaggagcaa acagcgtttg cgacatcctt ttgtaatact gcggaactga ctaaagtagt
     4381 gagttataca cagggctggg atctattctt tttatctttt tttattcttt ctttattcta
     4441 taaattataa ccacttgaat ataaacaaaa aaaacacaca aaggtctagc ggaatttaca
     4501 gagggtctag cagaatttac aagttttcca gcaaaggtct agcagaattt acagataccc
     4561 acaactcaaa ggaaaaggac tagtaattat cattgactag cccwtctcaa ttggtatagt
     4621 gattaaaatc acctagacca attgagatgt atgtctgaat tagttgtttt caaagcaaat
     4681 gaactagcga ttagtcgcta tgacttaacg gagcatgaaa ccaagctaat tttatgctgt
     4741 gtggcactac tcaaccccac gattgaaaac cctacaagga aagaacggac ggtatcgttc
     4801 acttataacc aatacgctca gatgatgaac atcagtaggg aaaatgctta tggtgtatta
     4861 gctaaagcaa ccagagagct gatgacgaga actgtggaaa tcaggaatcc tttggttaaa
     4921 ggctttaaga ttttccagtg gacaaactat gccaagttct caagcgaaaa attagaatta
     4981 gtttttagtg aagagatatt gccttatctt ttccagttaa aaaaattcat aaaatataat
     5041 ctggaacatg ttaagtcttt tgaaaacaaa tactctatga ggatttatga gtggttatta
     5101 aaagaactaa cacaaaagaa aactcacaag gcaaatatag agattagcct tgatgaattt
     5161 aagttcatgt taatgcttga aaataactac catgagttta aaaggcttaa ccaatgggtt
     5221 ttgaaaccaa taagtaaaga tttaaacact tacagcaata tgaaattggt ggttgataag
     5281 cgaggccgcc cgactgatac gttgattttc caagttgaac tagatagaca aatggatctc
     5341 gtaaccgaac ttgagaacaa ccagataaaa atgaatggtg acaaaatacc aacaaccatt
     5401 acatcagatt cctacctaca taacggacta agaaaaacac tacacgatgc tttaactgca
     5461 aaaattcagc tcaccagttt tgaggcaaaa tttttgagtg acatgcaaag taagtatgat
     5521 ctcaatggtt cgttctcatg gctcacgcaa aaacaacgaa ccacactaga gaacatactg
     5581 gctaaatacg gaaggatctg agtccatacg tcgcaaaaaa ccccgctttc ggcggggttt
     5641 tt
//
