LOCUS       sequence-391105-        5327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA position entry clone 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5327)
  AUTHORS   Lee RR, Cher WY, Wang J, Chen Y, Chae E
  TITLE     Generating minimum set of gRNA to cover multiple targets in multiple
            genomes with MINORg.
  JOURNAL   Nucleic Acids Res. 2023 Mar 15:gkad142. doi: 10.1093/nar/gkad142.
  PUBMED    36919598
REFERENCE   2  (bases 1 to 5327)
  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: "Nucleic 
            Acids Res. 2023 Mar 15:gkad142. doi: 10.1093/nar/gkad142."
COMMENT     Sequence Label: pECNUS_P2
FEATURES             Location/Qualifiers
     source          1..5327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(336..659)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     341..360
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     554..576
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     568..585
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     569..585
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    586..642
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(655..671)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(655..671)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(668..690)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    679..695
                     /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(703..709,710..727,728..733))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    748..769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    1030..1054
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1210..2358)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(2694..3282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2774..2793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3453..4244,4245..4313))
                     /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     4076..4095
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4314..4418)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4505..5136
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5228..5252
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tctgtgaaga caagcaagaa ttcaagcttg gagcatcttc attcttaaga tatgaagata
       61 atcttcaaaa ggcccctggg aatctgaaag aagagaagca ggcccattta tatgggaaag
      121 aacaatagta tttcttatat aggcccattt aagttgaaaa caatcttcaa aagtcccaca
      181 tcgcttagat aagaaaacga agctgagttt atatacagct agagtcgaag tagtgattgt
      241 gagaccgtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      301 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      361 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
      421 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      481 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      541 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt
      601 acccggggat cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata
      661 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
      721 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
      781 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcggtctc agtttaagag
      841 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
      901 tggcaccgag tcggtgcttt tttttgcaaa attttccaga tcgatttctt cttcctctgt
      961 tcttcggcgt tcaatttctg gggtcgctac tattgtcttc tgcacgaagt ggtttaaact
     1021 atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa
     1081 taatcggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg
     1141 ccaaccacag ggttccccag atcaggcgct ggctgctgaa cccccagccg gaactgaccc
     1201 cacaaggccc tagcgtttgc aatgcaccag gtcatcattg acccaggcgt gttccaccag
     1261 gccgctgcct cgcaactctt cgcaggcttc gccgacctgc tcgcgccact tcttcacgcg
     1321 ggtggaatcc gatccgcaca tgaggcggaa ggtttccagc ttgagcgggt acggctcccg
     1381 gtgcgagctg aaatagtcga acatccgtcg ggccgtcggc gacagcttgc ggtacttctc
     1441 ccatatgaat ttcgtgtagt ggtcgccagc aaacagcacg acgatttcct cgtcgatcag
     1501 gacctggcaa cgggacgttt tcttgccacg gtccaggacg cggaagcggt gcagcagcga
     1561 caccgattcc aggtgcccaa cgcggtcgga cgtgaagccc atcgccgtcg cctgtaggcg
     1621 cgacaggcat tcctcggcct tcgtgtaata ccggccattg atcgaccagc ccaggtcctg
     1681 gcaaagctcg tagaacgtga aggtgatcgg ctcgccgata ggggtgcgct tcgcgtactc
     1741 caacacctgc tgccacacca gttcgtcatc gtcggcccgc agctcgacgc cggtgtaggt
     1801 gatcttcacg tccttgttga cgtggaaaat gaccttgttt tgcagcgcct cgcgcgggat
     1861 tttcttgttg cgcgtggtga acagggcaga gcgggccgtg tcgtttggca tcgctcgcat
     1921 cgtgtccggc cacggcgcaa tatcgaacaa ggaaagctgc atttccttga tctgctgctt
     1981 cgtgtgtttc agcaacgcgg cctgcttggc ctcgctgacc tgttttgcca ggtcctcgcc
     2041 ggcggttttt cgcttcttgg tcgtcatagt tcctcgcgtg tcgatggtca tcgacttcgc
     2101 caaacctgcc gcctcctgtt caagacgacg cgaacgctcc acggcggccg atggcgcggg
     2161 cagggcaggg ggagccagtt gcacgctgtc gcgctcgatc ttggccgtag cttgctggac
     2221 catcgagccg acggactgga aggtttcgcg gggcgcacgc atgacggtgc ggcttgcgat
     2281 ggtttcggca tcctcggcgg aaaaccccgc gtcgatcagt tcttgcctgt atgccttccg
     2341 gtcaaacgtc cgattcattc accctccttg cgggattgcc ccgactcacg ccggggcaat
     2401 gtgcccttat tcctgatttg acccgcctgg tgccttggtg tccagataat ccaccttatc
     2461 ggcaatgaag tcggtcccgt agaccgtctg gccgtccttc tcgtacttgg tattccgaat
     2521 cttgccctgc acgaatacca gcgacccctt gcccaaatac ttgccgtggg cctcggcctg
     2581 agagccaaaa cacttgatgc ggaagaagtc ggtgcgctcc tgcttgtcgc cggcatcgtt
     2641 gcgccacatc taggatctgc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2701 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2761 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2821 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2881 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2941 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3001 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3061 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3121 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3181 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3241 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3301 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3361 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3421 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3481 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3541 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaacca
     3601 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3661 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3721 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3781 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3841 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3901 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3961 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4021 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4081 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4141 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4201 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4261 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4321 ctttttcaat attattgaag catttatcaa ggttattgtc tcatgagcgg atacatattt
     4381 gaatgtattt agaaaaataa acaaataggg gttccgcgca cgaattggcc agcgctgcca
     4441 tttttggggt gaggccgttc gcggccgagg ggcgcagccc ctggggggat gggaggcccg
     4501 cgttagcggg ccgggagggt tcgagaaggg ggggcacccc ccttcggcgt gcgcggtcac
     4561 gcgcacaggg cgcagccctg gttaaaaaca aggtttataa atattggttt aaaagcaggt
     4621 taaaagacag gttagcggtg gccgaaaaac gggcggaaac ccttgcaaat gctggatttt
     4681 ctgcctgtgg acagcccctc aaatgtcaat aggtgcgccc ctcatctgtc agcactctgc
     4741 ccctcaagtg tcaaggatcg cgcccctcat ctgtcagtag tcgcgcccct caagtgtcaa
     4801 taccgcaggg cacttatccc caggcttgtc cacatcatct gtgggaaact cgcgtaaaat
     4861 caggcgtttt cgccgatttg cgaggctggc cagctccacg tcgccggccg aaatcgagcc
     4921 tgcccctcat ctgtcaacgc cgcgccgggt gagtcggccc ctcaagtgtc aacgtccgcc
     4981 cctcatctgt cagtgagggc caagttttcc gcgaggtatc cacaacgccg gcggccgcgg
     5041 tgtctcgcac acggcttcga cggcgtttct ggcgcgtttg cagggccata gacggccgcc
     5101 agcccagcgg cgagggcaac cagcccggtg agcgtcgcaa aggagatcct gatctgactg
     5161 atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc
     5221 tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca
     5281 cattgcggac gtttttaatg tactggggtg gatgcagtgg gccccac
//
