LOCUS       sequence-363928-        3441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA cloning using blue-white screening for recombinants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3441)
  AUTHORS   Kozhukhar N, Spadafora D, Rodriguez YAR, Alexeyev MF
  TITLE     A Method for In Situ Reverse Genetic Analysis of Proteins Involved 
            mtDNA Replication.
  JOURNAL   Cells. 2022 Jul 11;11(14). pii: cells11142168. doi: 
            10.3390/cells11142168.
  PUBMED    35883613
REFERENCE   2  (bases 1 to 3441)
  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: "Cells."; 
            date: "2022-07-11"; volume: "11(14). pii: cells11142168. doi"; 
            pages: " 10.3390/cells11142168"
COMMENT     Sequence Label: pMA3735
FEATURES             Location/Qualifiers
     source          1..3441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    52..73
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(88..93,94..111,112..118)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    126..142
                     /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     150..166
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     185..205
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        187..205
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        343..591
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     343..363
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     514..533
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    705..726
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(741..746,747..764,765..771)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    779..795
                     /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     803..819
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             815..988
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
     primer_bind     complement(835..852)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(835..851)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(844..866)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_RNA        1002..1077
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1089..1113)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(1191..1209)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1192..1209)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1701..2495
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1755..1774)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2365..2384
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      2617..3205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3106..3125
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3359..3376
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
       61 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      121 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      181 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg
      241 ccagtgtgat ggatatctgc agaattcgcc ctttgtacaa aaaagcaggc tttaaaggaa
      301 ccaattcagt cgactggatc cggtaccaag gtcgggcagg aagagggcct atttcccatg
      361 attccttcat atttgcatat acgatacaag gctgttagag agataattag aattaatttg
      421 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
      481 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
      541 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgagagaccg
      601 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
      661 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      721 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      781 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgg attcactggc
      841 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
      901 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
      961 ccaacagttg cgcagcctga atggcgaatg gcgcggtctc agttttagag ctagaaatag
     1021 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     1081 ttttctagac ccagctttct tgtacaaagt tggcattaaa gggcgaattc cagcacactg
     1141 gcggccgtta ctagtggatc cgagctcggt accaagcttg atggccctat tctatagtgt
     1201 cacctaaatg ctagagatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1261 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgctcga
     1321 attgacgcgt cgaattaatt ccgctagctt cacgctgccg caagcactca gggcgcaagg
     1381 gctgctaaag gaagcggaac acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
     1441 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
     1501 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg
     1561 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
     1621 ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatct gatcaagaga
     1681 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     1741 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     1801 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     1861 ccggtgccct gaatgaactc caagacgagg cagcgcggct atcgtggctg gccacgacgg
     1921 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     1981 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     2041 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     2101 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     2161 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     2221 tcaaggcgcg gatgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     2281 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     2341 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     2401 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     2461 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgcggaatt
     2521 aattcgacgc gtcaattcga gcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2581 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2641 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2701 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2761 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2821 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2881 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2941 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3001 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3061 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3121 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3181 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3241 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3301 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3361 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     3421 ttggccgatt cattaatgca g
//
