LOCUS       sequence-271455-        8418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  obtaining full Cas12a RNP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8418)
  AUTHORS   Qiao J, Li W, Lin S, Sun W, Ma L, Liu Y
  TITLE     Co-expression of Cas9 and single-guided RNAs in Escherichia coli 
            streamlines production of Cas9 ribonucleoproteins.
  JOURNAL   Commun Biol. 2019 May 3;2:161. doi: 10.1038/s42003-019-0402-x. 
            eCollection 2019.
  PUBMED    31069270
REFERENCE   2  (bases 1 to 8418)
  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: "2019-05-3"; volume: "2:161. doi"; pages: " 
            10.1038/s42003-019-0402-x. eCollection 2019"
COMMENT     Sequence Label: pCold CL7-Cas12a
FEATURES             Location/Qualifiers
     source          1..8418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        48..114
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    117..133
                     /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)."
     5'UTR           155..288
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             292..309
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             703..726
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     3'UTR           4634..4778
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     primer_bind     complement(4819..4841)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4941..4960
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4983..5001
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        5022..5097
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5133..5204
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        5310..5414
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5415..5483,5484..6275)
                     /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     complement(5633..5652)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6446..7034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6935..6954
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7206..8288)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     8250..8269
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8289..8366)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgtggccg attaatcata
       61 aatatgaaaa ataattgttg catcacccgc caatgcgtgg cttaatgcac atcaaattgt
      121 gagcggataa caatttgatg tgctagcgca tatccagtgt agtaaggcaa gtcccttcaa
      181 gagttatcgt tgatacccct cgtagtgcac attcctttaa cgcttcaaaa tctgtaaagc
      241 acgccatatc gccgaaaggc acacttaatt attaagaggt aatacaccat gcaccatcat
      301 catcatcata tgagcaaaag caatgaaccg ggtaaagcaa ccggtgaagg taaaccggtt
      361 aataacaaat ggctgaacaa tgccggtaaa gatctgggta gtccggttcc ggatcgtatt
      421 gcaaataaac tgcgtgataa agaattcgag agcttcgatg attttcgtga aaccttttgg
      481 gaagaagtta gcaaagatcc tgaactgagc aaacagttta gccgcaataa caatgatcgt
      541 atgaaagttg gtaaagcacc gaaaacacgt acccaggatg ttagcggtaa acgtacctca
      601 tttgaactga atcatcagaa accgattgaa cagaatggtg gcgtttatga tatggataac
      661 attagcgttg ttaccccgaa acgcaacatt gatattgaag gcttggaggt tttgttccag
      721 ggtccaatgt caatttatca agaatttgtg aacaaatata gcctgagcaa aaccctgcgt
      781 tttgaactga ttccgcaggg taaaaccctg gaaaacatta aagcacgtgg tctgattctg
      841 gatgatgaaa aacgtgccaa agactacaaa aaagccaaac aaatcatcga taaataccac
      901 cagttcttca tcgaagaaat tctgagcagc gtttgcatta gcgaagatct gctgcagaat
      961 tattccgacg tttatttcaa actgaaaaaa agcgacgatg ataacctgca gaaagatttc
     1021 aaaagcgcca aagataccat caaaaaacaa attagcgagt atatcaaaga cagcgagaaa
     1081 ttcaaaaacc tgttcaacca gaatctgatc gatgccaaaa aaggtcaaga aagcgatctg
     1141 atcctgtggc tgaaacagag caaagataat ggcatcgaac tgtttaaagc caacagcgat
     1201 attaccgata ttgatgaagc actggaaatc atcaaaagct ttaaaggttg gaccacctac
     1261 tttaaaggct ttcacgaaaa tcgcaaaaac gtgtatagca gcaatgatat tccgaccagc
     1321 attatctatc gcatcgttga tgataatctg cctaaatttc tggaaaataa agccaaatat
     1381 gaaagcctga aagacaaagc accggaagca attaactatg agcagatcaa aaaagatctg
     1441 gccgaagaac tgacctttga cattgattac aaaaccagcg aagttaacca gcgtgttttt
     1501 agcctggatg aagtttttga aattgccaac ttcaacaact acctgaatca gagcggtatc
     1561 accaaattca ataccattat cggtggcaaa ttcgtgaatg gcgaaaatac caaacgcaaa
     1621 ggcatcaacg aatacattaa tctgtatagc cagcagatta acgataaaac gctgaaaaaa
     1681 tacaaaatga gcgtgctgtt caaacaaatt ctgtcagata ccgaaagcaa aagcttcgtg
     1741 attgacaaac tggaagatga tagtgatgtt gttaccacca tgcagagctt ttatgaacaa
     1801 atcgcagcgt ttaaaaccgt ggaagagaaa tccattaaag aaaccctgag cctgctgttt
     1861 gatgatctga aagcacagaa actggacctg tccaaaatct acttcaaaaa cgataaatcc
     1921 ctgaccgatc tgagccagca ggttttcgat gattatagcg ttattggcac cgcagttctg
     1981 gaatatatca cacagcagat tgcaccgaaa aatctggata atccgagcaa aaaagaacaa
     2041 gagctgatcg ccaaaaaaac cgagaaagcg aaatatctga gcctggaaac aattaaactg
     2101 gccctggaag aatttaacaa acaccgcgac attgataaac agtgccgctt tgaagaaatc
     2161 ctggcaaatt ttgcagcaat cccgatgatc tttgatgaaa ttgcgcagaa taaagataac
     2221 ctggcacaga tcagcatcaa atatcagaat cagggaaaaa aagacctgct gcaagcaagt
     2281 gccgaagatg atgttaaagc gattaaagat ctgctggatc agaccaataa cctgctgcat
     2341 aaactgaaaa tctttcacat tagccagagc gaggataaag cgaacattct ggataaagat
     2401 gagcacttct atctggtgtt tgaagagtgt tattttgagc tggcaaatat tgtgccgctg
     2461 tataacaaaa tccgcaacta tattacccag aaaccgtata gcgacgaaaa attcaaactg
     2521 aactttgaga atagcaccct ggccaatggt tgggataaaa acaaagaacc ggataatacc
     2581 gccatcctgt tcattaaaga tgataaatac tatctgggcg tgatgaacaa aaaaaacaac
     2641 aaaatcttcg acgataaagc catcaaagag aataaaggcg aaggttacaa aaaaatcgtg
     2701 tacaaactgc tgcctggtgc gaataaaatg ctgccgaaag tgttttttag cgccaaatcc
     2761 atcaaattct ataacccgag cgaagatatt ctgcgtattc gtaatcatag cacccatacc
     2821 aaaaatggta gtccgcagaa aggctatgaa aaattcgagt tcaacattga ggattgccgc
     2881 aaattcatcg acttctacaa acagtccatt agcaaacatc cggaatggaa agactttggt
     2941 tttcgtttta gcgataccca gcgctataac agcattgatg aattttatcg cgaagtggaa
     3001 aaccagggct ataaactgac atttgaaaac atcagcgaga gctatattga tagcgttgtg
     3061 aatcagggta aactgtacct gtttcagatc tataacaaag actttagcgc ctatagcaaa
     3121 ggtcgtccga atctgcatac cctgtattgg aaagcactgt tcgatgaacg taatctgcag
     3181 gatgttgtct acaaactgaa tggtgaagca gaactgtttt atcgcaaaca gagtatcccg
     3241 aaaaaaatca cccatccggc aaaagaagca atcgcgaaca aaaacaaaga taacccgaaa
     3301 aaagaaagcg tgttcgagta tgatctgatc aaagataaac gcttcaccga agataaattc
     3361 tttttccatt gcccgatcac catcaacttt aaaagcagcg gtgcgaacaa attcaacgat
     3421 gaaatcaatc tgctgctgaa agaaaaagcc aacgatgttc atattctgag cattgatcgt
     3481 ggtgaacgtc atctggccta ttacaccctg gttgatggta aaggcaatat tatcaaacag
     3541 gacaccttca acattatcgg caatgatcgt atgaaaacca actaccatga taaactggca
     3601 gccattgaaa aagatcgtga tagcgcacgt aaagattgga aaaaaatcaa caacattaaa
     3661 gaaatgaaag aaggctacct gagccaggtt gttcatgaaa tcgccaaact ggtgattgaa
     3721 tataatgcca ttgtggtgtt cgaggatctg aacttcggtt tcaaacgtgg tcgtttcaaa
     3781 gttgagaaac aggtgtatca aaaactggaa aaaatgctga tcgaaaaact gaattacctg
     3841 gtgttcaaag acaacgaatt cgataaaacc ggtggtgttc tgcgtgcata tcagctgacc
     3901 gcaccttttg aaaccttcaa aaaaatgggt aaacagaccg gcatcatcta ttatgttccg
     3961 gcaggtttta cctccaaaat ttgtccggtt accggctttg ttaatcagct gtatccgaaa
     4021 tatgagagcg ttagcaaaag ccaagagttt ttcagcaaat ttgataaaat ctgctataac
     4081 ctggacaaag gctactttga attcagcttt gactataaaa actttggcga taaagcagcc
     4141 aaaggcaaat ggaccattgc aagctttggt agccgtctga ttaactttcg taacagcgac
     4201 aaaaaccata actgggatac ccgtgaagtt tatccgacca aagagctgga aaaactgctg
     4261 aaagattaca gcattgaata tggtcatggc gaatgtatta aagccgcaat ttgtggtgag
     4321 tccgacaaaa aattctttgc aaaactgacc agcgtgctga ataccattct gcagatgcgt
     4381 aatagcaaaa ccggcaccga actggattat ctgattagtc cggttgcaga tgtgaacggc
     4441 aattttttcg atagccgtca ggctccgaaa aatatgccgc aggatgcaga tgcaaatggt
     4501 gcctatcata ttggcctgaa aggtctgatg ctgctgggtc gcattaaaaa caatcaagaa
     4561 ggcaaaaaac tgaacctggt gatcaaaaac gaagagtatt ttgagttcgt gcagaatagg
     4621 aataactagg taatctctgc ttaaaagcac agaatctaag atccctgcca tttggcgggg
     4681 atttttttat ttgttttcag gaaataaata atcgatcgcg taataaaatc tattattatt
     4741 tttgtgaaga ataaatttgg gtgcaatgag aatgcgcagg ccctttcgtc tcgcgcgttt
     4801 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4861 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4921 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagtcg
     4981 actaatacga ctcactatag gtgacatcaa ttattataca tgttttagag ctagaaatag
     5041 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgctta
     5101 gcatccaaac tcgagtaagg atctccaggc atcaaataaa acgaaaggct cagtcgaaag
     5161 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcgtcgac actatggttg
     5221 ctttgacgta tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     5281 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5341 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5401 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5461 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5521 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5581 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5641 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5701 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5761 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5821 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5881 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5941 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6001 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6061 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6121 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6181 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6241 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6301 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6361 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6421 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6481 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6541 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6601 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6661 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6721 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6781 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6841 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6901 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6961 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7021 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7081 cttttgctca catagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     7141 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     7201 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     7261 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     7321 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     7381 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     7441 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     7501 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     7561 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     7621 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     7681 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     7741 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     7801 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     7861 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     7921 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     7981 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     8041 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     8101 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     8161 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     8221 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     8281 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     8341 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     8401 cattaggaag cagcccag
//
