LOCUS       sequence-364202-        5482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chromosomal transfer helper plasmid with sgRNAs (one fixed, one 
            flexible) for in-vivo excision of transferred DNA for homologous 
            recombination and counter-selection for successful integration..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5482)
  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 5482)
  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: pDrop
FEATURES             Location/Qualifiers
     source          1..5482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             154..459
                     /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     338..357
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        567..642
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     RBS             849..860
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      1571..1642
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1658..1685
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(1696..2487,2488..2556))
                     /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     2319..2338
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2557..2661)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    2764..2782
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2789..2807
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        2838..2913
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2933..3003
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      3455..3677
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             3725..4675
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     protein_bind    4734..4752
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4759..4777
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4861..4882
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4897..4902,4903..4920,4921..4927)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4935..4951
                     /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     4940..4962
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4959..4975
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4959..4975
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    4988..5044
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(5045..5062)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5045..5061)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5054..5076)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5270..5289)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
       61 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
      121 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
      181 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
      241 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
      301 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
      361 agtgtgcctg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
      421 atcaaaaacg ccattaacct gatgttctgg ggaatataat taagccagcc ccgacacccg
      481 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
      541 gctgtgaccc actcaagtgg gtctcagttt tagagctaga aatagcaagt taaaataagg
      601 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttta cggtcatgta
      661 aaaaaaaacc ccgcccctga cagggcgggg ttttttttca gtcaagcctc tcttgacatc
      721 gctagaaatt gtcttaagat atgttgctta tttaatgaaa cggcaggttc tcttgacatc
      781 gctagaaatt gtcttaagat atgttgctta tttaatgaaa cggcaggttc ggatcttagc
      841 tactagagaa agaggagaaa tactagatgc gtaaaggcga agcagtgatc aaagaattta
      901 tgcgcttcaa agttcacatg gaaggttcta tgaacggcca cgaatttgaa attgaaggtg
      961 aaggcgaagg tcgtccatac gaaggtactc aaaccgcaaa actgaaagtt accaaaggtg
     1021 gtccactgcc attttcttgg gatattctgt ctccacaatt tatgtacggt tctcgtgcat
     1081 ttatcaaaca cccagcagat attccagact actacaaaca atcttttccg gaaggtttca
     1141 aatgggaacg tgttatgaat tttgaagatg gtggtgcagt tacggttacc caagatacct
     1201 ctctggaaga tggtactctg atctacaaag ttaaactgcg tggtactaac tttccaccag
     1261 atggtccagt tatgcagaaa aaaaccatgg gttgggaagc atctaccgaa cgtctgtacc
     1321 cagaagatgg cgttctgaaa ggtgatatca aaatggcact gcgtctgaaa gatggcggtc
     1381 gttacctggc agatttcaaa accacctaca aagcgaaaaa accagttcaa atgccaggtg
     1441 catacaacgt tgatcgtaaa ctggatatta ccagccacaa cgaagattac accgttgttg
     1501 aacaatacga acgttctgaa ggccgtcact ctaccggtgg tatggatgaa ctgtacaaat
     1561 aaccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     1621 ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt
     1681 ctgcgtttat aatgattacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     1741 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     1801 cttaccatct ggccccagtg ctgcaatgat accgcgagaa ccacgctcac cggctccaga
     1861 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1921 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1981 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2041 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2101 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2161 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2221 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2281 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2341 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     2401 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     2461 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     2521 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     2581 aagcatttat caaggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2641 taaacaaata ggggttccgc gcaactccgg caggacttgt tcagaacgct cggtcttgca
     2701 caccgggcgt tttttctttg tgagtccagg caagctttcg ctaaggatga tttctggaat
     2761 tcttccctat cagtgataga gattgacatc cctatcagtg atagagatac tgagcacgct
     2821 cgatcgctaa tcgttatgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     2881 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt caagccaggc atcaaataaa
     2941 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     3001 tctgggtgca ttagccagtc tgaatgacct gtcacgggat aatccgaagt ggtcagactg
     3061 gaaaatcaga gggcaggaac tgctgaacag caaaaagtca gatagcacca catagcagac
     3121 ccgccataaa acgccctgag aagcccgtga cgggcttttc ttgtattatg ggtagtttcc
     3181 ttgcatgaat ccataaaagg cgcctgtagt gccatttacc cccattcact gccagagccg
     3241 tgagcgcagc gaactgaatg tcacgaaaaa gacagcgact caggtgcctg atggtcggag
     3301 acaaaaggaa tattcagcga tttgcccgat tgcggccgca accgagcttg cgagggtgct
     3361 acttaagcct ttagggtttt aaggtctgtt ttgtagagga gcaaacagcg tttgcgacat
     3421 ccttttgtaa tactgcggaa ctgactaaag tagtgagtta tacacagggc tgggatctat
     3481 tctttttatc tttttttatt ctttctttat tctataaatt ataaccactt gaatataaac
     3541 aaaaaaaaca cacaaaggtc tagcggaatt tacagagggt ctagcagaat ttacaagttt
     3601 tccagcaaag gtctagcaga atttacagat acccacaact caaaggaaaa ggactagtaa
     3661 ttatcattga ctagcccatc tcaattggta tagtgattaa aatcacctag accaattgag
     3721 atgtatgtct gaattagttg ttttcaaagc aaatgaacta gcgattagtc gctatgactt
     3781 aacggagcat gaaaccaagc taattttatg ctgtgtggca ctactcaacc ccacgattga
     3841 aaaccctaca aggaaagaac ggacggtatc gttcacttat aaccaatacg ctcagatgat
     3901 gaacatcagt agggaaaatg cttatggtgt attagctaaa gcaaccagag agctgatgac
     3961 gagaactgtg gaaatcagga atcctttggt taaaggcttt gagattttcc agtggacaaa
     4021 ctatgccaag ttctcaagcg aaaaattaga attagttttt agtgaagaga tattgcctta
     4081 tcttttccag ttaaaaaaat tcataaaata taatctggaa catgttaagt cttttgaaaa
     4141 caaatactct atgaggattt atgagtggtt attaaaagaa ctaacacaaa agaaaactca
     4201 caaggcaaat atagagatta gccttgatga atttaagttc atgttaatgc ttgaaaataa
     4261 ctaccatgag tttaaaaggc ttaaccaatg ggttttgaaa ccaataagta aagatttaaa
     4321 cacttacagc aatatgaaat tggtggttga taagcgaggc cgcccgactg atacgttgat
     4381 tttccaagtt gaactagata gacaaatgga tctcgtaacc gaacttgaga acaaccagat
     4441 aaaaatgaat ggtgacaaaa taccaacaac cattacatca gattcctacc tacataacgg
     4501 actaagaaaa acactacacg atgctttaac tgcaaaaatt cagctcacca gttttgaggc
     4561 aaaatttttg agtgacatgc aaagtaagta tgatctcaat ggttcgttct catggctcac
     4621 gcaaaaacaa cgaaccacac tagagaacat actggctaaa tacggaagga tctgagtcca
     4681 tacgtcgcaa aaaaccccgc tcggcggggt tttttcgctg tagcacctga agttccctat
     4741 cagtgataga gattgacatc cctatcagtg atagagatac tgagcactga gacccagtgg
     4801 tatgggcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     4861 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     4921 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     4981 ttacgccaag cttgcatgcc tgcaggtcga ctctagagga tccccgggta ccgagctcga
     5041 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     5101 atcgccttgc aggacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     5161 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     5221 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     5281 ctgatgccgc atagtcgcgt ggatccggct tactaaaagc cagataacag tatgcgtatt
     5341 tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa
     5401 aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt
     5461 gctcaaggca tatatgatgt ca
//
