LOCUS       sequence-449467-        6362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Prokaryotic and constitutive expression of AsCas12f1/APOBEC1-LVA 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6362)
  AUTHORS   van der Oost
  TITLE     van der Oost Lab Plasmids 2024
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6362)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pdCas12f1_CBE-LVA
FEATURES             Location/Qualifiers
     source          1..6362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      211..666
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(298..317)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     508..529
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1222..1881)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGVA"
     primer_bind     1796..1815
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1882..1984)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2376..2393)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2510..3055)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2590..2609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3131..3150
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3245..3279
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     CDS             4611..5297
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             5298..5318
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5331..5579
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     primer_bind     complement(5630..5647)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5800..5886
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5978..6005
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        6024..6115
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
       61 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
      121 aaatagacag atcgctgaga taggtgcctc actgattaag cattgataac tgtcagacca
      181 agtttactca tatatacttt agattgattt acgcgccctg tagcggcgca ttaagcgcgg
      241 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      301 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      361 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      421 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      481 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      541 accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt
      601 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
      661 caatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
      721 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
      781 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
      841 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
      901 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
      961 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt caggcatttg
     1021 agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat
     1081 aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt
     1141 tctgccattc atccgcttat tatcacttat tcaggcgtag caaccaggcg tttaagggca
     1201 ccaataactg ccttaaaaaa attacgccac gccctgccac tcatcgcagt actgttgtaa
     1261 ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc
     1321 cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc
     1381 gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt
     1441 ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc
     1501 gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc
     1561 actccagagc gatgaaaacg tttcagtttg ctcatgaaaa acggtgtaac aagggtgaac
     1621 actatcccat atcaccagct caccgtcttt cattgccata cggaattccg gatgagcatt
     1681 catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac
     1741 ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac
     1801 tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata
     1861 tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa
     1921 aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg
     1981 atcaacgtct cattttcgcc aaaagttggc ccagggcttc ccggtatcaa catggacacc
     2041 aggatttatt tattctgcga agtgatcttc cgtcacaggt atttattcgg cgcaaagtgc
     2101 gtcgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga ggtgctccag
     2161 tggcttctgt ttctatcagc tgtccctcct gttcagctac tgacggggtg gtgcgtaacg
     2221 gcaaaagcac cgccggacat cagcgctagc ggagtgtata ctggcttact atgttggcac
     2281 tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt
     2341 cagcagaata tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg
     2401 gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcgaagattt cctggaagat
     2461 gccaggaaga tacttaacag ggaagtaaga gggccgcggc aaagccgttt ttccataggc
     2521 tccgcccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga
     2581 caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc
     2641 ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct cattccacgc
     2701 ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc
     2761 gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga
     2821 catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa
     2881 gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag
     2941 ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa
     3001 ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga
     3061 tcatcttatt aatcagataa aatatttcta ggctcatgag cccgaagtgg cgagcccgat
     3121 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgctggtga
     3181 tgccggccac aatgcgtccg gcgtagagga tctgctcatg tttgacagct tatcatcgat
     3241 gcatctgaca gctagctcag tcctaggtat aatgctagcg agctcaggag gacatcatga
     3301 tcaaagttta ccgttatgaa atcgttaaac cgctggacct ggattggaaa gaattcggca
     3361 ccatcctgcg tcagctgcag caggaaaccc gcttcgcgct gaacaaagca acccagctgg
     3421 cgtgggaatg gatgggtttc agctctgatt acaaagacaa ccatggcgaa tacccgaaaa
     3481 gcaaagacat cctgggctac accaacgttc acggttacgc ataccacacc atcaaaacca
     3541 aagcttatcg tctgaacagc ggcaacctga gccagaccat caaacgtgcg accgatcgct
     3601 tcaaagcgta ccagaaagaa atcctgcgtg gtgacatgtc catcccgtct tacaaacgtg
     3661 atatcccgct ggatctgatc aaagaaaaca tcagcgttaa ccgtatgaac cacggtgatt
     3721 atatcgcttc cctgtccctg ctgtctaacc cggcgaaaca ggaaatgaac gttaaacgta
     3781 aaatctccgt gattatcatc gttcgtggcg cgggtaaaac catcatggat cgcatcctga
     3841 gcggcgaata ccaggtgagc gcgagccaga tcatccacga cgaccgtaaa aacaaatggt
     3901 acctgaacat ctcttatgat ttcgaaccgc agacccgtgt tctggatctg aacaaaatca
     3961 tgggcatcgc tctgggtgtt gcggtggcgg tgtacatggc gttccagcac accccggcgc
     4021 gctacaaact ggaaggcggt gaaatcgaaa acttccgtcg tcaggtggag tctcgtcgca
     4081 tctccatgct gcgccagggt aaatacgcgg gtggtgctcg tggcggtcac ggccgtgata
     4141 aacgtatcaa accgatcgaa cagctgcgtg acaaaatcgc taacttccgc gataccacta
     4201 accaccgtta ctctcgttac atcgttgata tggctatcaa agaaggttgc ggtaccattc
     4261 agatggaaga tctgaccaac atccgtgata tcggctctcg tttcctgcag aactggacct
     4321 actacgatct gcagcagaaa atcatctaca aagcggaaga agcgggcatc aaagttatca
     4381 aaattgatcc gcagtacacc agccagcgtt gcagcgaatg cggcaacatc gattccggta
     4441 accgtatcgg tcaggcgatc ttcaaatgcc gtgcttgcgg ctacgaagcg aacgcggact
     4501 acaacgcagc tcgtaacatc gcgatcccga acatcgataa aatcatcgcg gaaagcatca
     4561 aaagcggcag cgagactccc gggacctcag agtccgccac acccgaaagt atgagctcag
     4621 agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc catgagtttg
     4681 aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac gaaattaatt
     4741 gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag cacgtcgaag
     4801 tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca aggtgcagca
     4861 ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc actgaattcc
     4921 tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac caccacgctg
     4981 acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact atccaaatta
     5041 tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc ccgagtaatg
     5101 aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt gaactgtact
     5161 gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag ccacagctga
     5221 cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca cacattctct
     5281 gggccaccgg gttgaaaccc aagaagaaac ggaaagtgtc tggtggttct actaatctgt
     5341 cagatattat tgaaaaggag accggtaagc aactggttat ccaggaatcc atcctcatgc
     5401 tcccagagga ggtggaagaa gtcattggga acaagccgga aagcgatata ctcgtgcaca
     5461 ccgcctacga cgagagcacc gacgagaatg tcatgcttct gactagcgac gcccctgaat
     5521 acaagccttg ggctctggtc atacaggata gcaacggtga gaacaagatt aagatgctct
     5581 tagtagctta aaagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga
     5641 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     5701 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     5761 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     5821 tcaaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     5881 acaaatccgc cgggagcgga tttgaacgtt gcaaagcaac ggcccggagg gtggcgggca
     5941 aaacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     6001 ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc
     6061 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6121 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6181 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcagcaaa
     6241 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6301 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6361 ga
//
