LOCUS       sequence-357155-        3514 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA Position Vector 4, used to create multi gRNA NT-CRISPR plasmid 
            with pST_119_LVL2 cam.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3514)
  AUTHORS   Stukenberg D, Hoff J, Faber A, Becker A
  TITLE     NT-CRISPR, combining natural transformation and CRISPR-Cas9 
            counterselection for markerless and scarless genome editing in 
            Vibrio natriegens.
  JOURNAL   Commun Biol. 2022 Mar 25;5(1):265. doi: 10.1038/s42003-022-03150-0.
  PUBMED    35338236
REFERENCE   2  (bases 1 to 3514)
  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: "Commun 
            Biol."; date: "2022-03-25"; volume: "5(1):265. doi"; pages: " 
            10.1038/s42003-022-03150-0"
COMMENT     Sequence Label: pMC0x_gRNA_Pos4_Ptet
FEATURES             Location/Qualifiers
     source          1..3514
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    49..67
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     RBS             234..245
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             252..965
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      977..1048
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1064..1091
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      1319..1376
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(1400..1417)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1571..2159)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1651..1670)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2392..3207)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2392..3207)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3115..3134
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3199..3218
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3412..3430
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3434..3477)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3480..3501
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
ORIGIN
        1 ctcggcttca aatcactgta atgatcttaa ttcctaattt ttgttgacac tctatcattg
       61 atagagttat tttagtccag agaccgaaag tgaaacgtga tttcatgcgt cattttgaac
      121 attttgtaaa tcttatttaa taatgtgtgc ggcaattcac atttaattta tgaatgtttt
      181 cttaacatcg cggcaactca agaaacggca ggttcggatc ttagctacta gagaaagagg
      241 agaaatacta gatgcgtaaa ggcgaagaac ttttcactgg cgtagtacca attttagtgg
      301 agctggacgg agatgtaaat ggtcataagt tttcagttcg aggagaaggc gaaggagatg
      361 caactaacgg taagctgaca ctaaagttta tctgtaccac gggtaaactg ccggtcccat
      421 ggccgacact ggttactaca cttacttatg gtgtacaatg ctttgctcgt tatcctgacc
      481 atatgaagca gcacgatttc ttcaaatcgg ccatgcctga aggatacgtg caagaacgta
      541 caattagctt taaagatgac ggcacctata aaacgcgcgc cgaagttaaa ttcgagggtg
      601 acacattggt taatcgtata gaacttaagg gcattgactt caaagaagat ggcaacatcc
      661 ttggccataa actggaatat aattttaact ctcacaatgt ctacattacg gcggataaac
      721 aaaagaatgg cattaaagcg aatttcaaaa tccgccacaa tgtcgaagac ggctcggttc
      781 aactggcgga ccattatcag cagaacaccc caatcggtga tggcccggtt ctgttacctg
      841 ataatcatta cctttctact caaagcgttt tatctaaaga tcctaacgag aagcgtgatc
      901 atatggttct actggaattt gttaccgcag ctggtatcac gcacggcatg gatgagctgt
      961 ataaataacc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1021 tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg
     1081 gcctttctgc gtttataggt ctcagtttca gagctatgct ggaaacagca tagcaagttg
     1141 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttctcg
     1201 gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt ttggtccacc
     1261 ctgcacgata acactgaggt atgagacgac tcatcgcaac tagtagcggc cgctgcagtc
     1321 cggcaaaaaa gggcaaggtg tcaccaccct gccctttttc tttaaaaccg aaaagattac
     1381 ttcgcgttat gcaggcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1441 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1501 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1561 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1621 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1681 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     1741 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     1801 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     1861 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     1921 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     1981 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2041 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2101 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2161 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2221 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     2281 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagc tcgagtcccg
     2341 tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa
     2401 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2461 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2521 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     2581 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2641 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2701 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2761 cagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2821 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2881 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2941 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     3001 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     3061 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     3121 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca
     3181 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     3241 cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg
     3301 agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagctcga
     3361 gtgccacttg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     3421 atcacgaggc agaatttcag ataaaaaaaa tccttagctt tcgctaagga tgatttctgg
     3481 aattcgcggc cgcttctaga gagcgatgag tcgt
//
