LOCUS       sequence-344533-        7288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vivo Cre-lox circularization in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7288)
  AUTHORS   Enghiad B, Huang C, Guo F, Jiang G, Wang B, Tabatabaei SK, Martin 
            TA, Zhao H
  TITLE     Cas12a-assisted precise targeted cloning using in vivo Cre-lox 
            recombination.
  JOURNAL   Nat Commun. 2021 Feb 19;12(1):1171. doi: 10.1038/s41467-021-21275-4.
  PUBMED    33608525
REFERENCE   2  (bases 1 to 7288)
  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: "Nat 
            Commun."; date: "2021-02-19"; volume: "12(1):1171. doi"; pages: " 
            10.1038/s41467-021-21275-4"
COMMENT     Sequence Label: pBE14
FEATURES             Location/Qualifiers
     source          1..7288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(219..1409)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTITLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     1038..1057
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(1457..1462,1463..1479,1480..1485))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             complement(1530..2408)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2435..2719
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     2642..2661
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             2746..2754
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             2763..3179
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the 
                     lambda Red system"
                     /label=Gam
                     /note="protects the double-stranded DNA substrate for 
                     recombination"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     RBS             3180..3191
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3192..4253
                     /codon_start=1
                     /gene="recA"
                     /product="DNA repair protein from E. coli"
                     /label=RecA
                     /translation="MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGS
                     LSLDIALGAGGLPMGRIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY
                     ARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIDDS
                     HMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASV
                     RLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVK
                     EKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSV
                     DDSEGVAETNEDF"
     terminator      4254..4498
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(4621..5571)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      5619..5841
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     promoter        6095..6129
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             6130..6141
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             6142..7173
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     terminator      7174..7208
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
ORIGIN
        1 ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt
       61 ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag
      121 taaggcagcg gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgtcttgg
      181 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
      241 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
      301 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
      361 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
      421 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
      481 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
      541 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
      601 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
      661 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
      721 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
      781 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
      841 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
      901 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
      961 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1021 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1081 ccggtgatgc cggccacgat gcgtccggcg tagaggatcc acaggacggg tgtggtcgcc
     1141 atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg gcggcggcca
     1201 aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa cgcatatagc
     1261 gctagcagca cgccatagtg actggcgatg ctgtcggaat ggacgatatc ccgcaagagg
     1321 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgat ggtgccgagg
     1381 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     1441 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agacacattt
     1501 ccccgaaaag tgccacctgc atcgatttat tatgacaact tgacggctac atcattcact
     1561 ttttcttcac aaccggcacg gaactcgctc gggctggccc cggtgcattt tttaaatacc
     1621 cgcgagaagt agagttgatc gtcaaaacca acattgcgac cgacggtggc gataggcatc
     1681 cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag
     1741 acgctaatcc ctaactgctg gcggaaaaga tgtgacagac gcgacggcga caagcaaaca
     1801 tgctgtgcga cgctggcgat atcaaaattg ctgtctgcca ggtgatcgct gatgtactga
     1861 caagcctcgc gtacccgatt atccatcggt ggatggagcg actcgttaat cgcttccatg
     1921 cgccgcagta acaattgctc aagcagattt atcgccagca gctccgaata gcgcccttcc
     1981 ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca
     2041 tccgggcgaa agaaccccgt attggcaaat attgacggcc agttaagcca ttcatgccag
     2101 taggcgcgcg gacgaaagta aacccactgg tgataccatt cgcgagcctc cggatgacga
     2161 ccgtagtgat gaatctctcc tggcgggaac agcaaaatat cacccggtcg gcaaacaaat
     2221 tctcgtccct gatttttcac caccccctga ccgcgaatgg tgagattgag aatataacct
     2281 ttcattccca gcggtcggtc gataaaaaaa tcgagataac cgttggcctc aatcggcgtt
     2341 aaacccgcca ccagatgggc attaaacgag tatcccggca gcaggggatc attttgcgct
     2401 tcagccatac ttttcatact cccgccattc agagaagaaa ccaattgtcc atattgcatc
     2461 agacattgcc gtcactgcgt cttttactgg ctcttctcgc taaccaaacc ggtaaccccg
     2521 cttattaaaa gcattctgta acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa
     2581 agtgtctata atcacggcag aaaagtccac attgattatt tgcacggcgt cacactttgc
     2641 tatgccatag catttttatc cataagatta gcggatccta cctgacgctt tttatcgcaa
     2701 ctctctactg tttctccata cccgtttttt tgggaattcg agctctaagg aggttataaa
     2761 aaatggatat taatactgaa actgagatca agcaaaagca ttcactaacc ccctttcctg
     2821 ttttcctaat cagcccggca tttcgcgggc gatattttca cagctatttc aggagttcag
     2881 ccatgaacgc ttattacatt caggatcgtc ttgaggctca gagctgggcg cgtcactacc
     2941 agcagctcgc ccgtgaagag aaagaggcag aactggcaga cgacatggaa aaaggcctgc
     3001 cccagcacct gtttgaatcg ctatgcatcg atcatttgca acgccacggg gccagcaaaa
     3061 aatccattac ccgtgcgttt gatgacgatg ttgagtttca ggagcgcatg gcagaacaca
     3121 tccggtacat ggttgaaacc attgctcacc accaggttga tattgattca gaggtataaa
     3181 aagaggagaa aatggctatc gacgaaaaca aacagaaagc gttggcggca gcactgggcc
     3241 agattgagaa acaatttggt aaaggctcca tcatgcgcct gggtgaagac cgttccatgg
     3301 atgtggaaac catctctacc ggttcgcttt cactggatat cgcgcttggg gcaggtggtc
     3361 tgccgatggg ccgtatcgtc gaaatctacg gaccggaatc ttccggtaaa accacgctga
     3421 cgctgcaggt gatcgccgca gcgcagcgtg aaggtaaaac ctgtgcgttt atcgatgctg
     3481 aacacgcgct ggacccaatc tacgcacgta aactgggcgt cgatatcgac aacctgctgt
     3541 gctcccagcc ggacaccggc gagcaggcac tggaaatctg tgacgccctg gcgcgttctg
     3601 gcgcagtaga cgttatcgtc gttgactccg tggcggcact gacgccgaaa gcggaaatcg
     3661 aaggcgaaat cgacgactct cacatgggcc ttgcggcacg tatgatgagc caggcgatgc
     3721 gtaagctggc gggtaacctg aagcagtcca acacgctgct gatcttcatc aaccagatcc
     3781 gtatgaaaat tggtgtgatg ttcggtaacc cggaaaccac taccggtggt aacgcgctga
     3841 aattctacgc ctctgttcgt ctcgacatcc gtcgtatcgg cgcggtgaaa gagggcgaaa
     3901 acgtggtggg tagcgaaacc cgcgtgaaag tggtgaagaa caaaatcgct gcgccgttta
     3961 aacaggctga attccagatc ctctacggcg aaggtatcaa cttctacggc gaactggttg
     4021 acctgggcgt aaaagagaag ctgatcgaga aagcaggcgc gtggtacagc tacaaaggtg
     4081 agaagatcgg tcagggtaaa gcgaatgcga ctgcctggct gaaagataac ccggaaaccg
     4141 cgaaagagat cgagaagaaa gtacgtgagt tgctgctgag caacccgaac tcaacgccgg
     4201 atttctctgt agatgatagc gaaggcgtag cagaaactaa cgaagatttt taacgcatcc
     4261 tcacgataat atccgggtag gcgcaatcac tttcgtctac tccgttacaa agcgaggctg
     4321 ggtatttccc ggcctttctg ttatccgaaa tccactgaaa gcacagcggc tggctgagga
     4381 gataaataat aaacgagggg ctgtatgcac aaagcatctt ctgttgagtt aagaacgagt
     4441 atcgagatgg cacatagcct tgctcaaatt ggaatcaggt ttgtgccaat accagtagaa
     4501 acagacgaag aatccatggg tatggacagt tttccctttg atatgtaacg gtgaacagtt
     4561 gttctacttt tgtttgttag tcttgatgct tcactgatag atacaagagc cataagaacc
     4621 tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag
     4681 ccatgagaac gaaccattga gatcatactt actttgcatg tcactcaaaa attttgcctc
     4741 aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt
     4801 atgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg
     4861 gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa
     4921 cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa
     4981 atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt
     5041 ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt
     5101 tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc
     5161 aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg
     5221 caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt
     5281 ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat
     5341 cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat
     5401 ctgaacgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat
     5461 cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc
     5521 atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa
     5581 ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat gataattact
     5641 agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact
     5701 tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta
     5761 tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga
     5821 tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc
     5881 gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac
     5941 cctcgcaagc tcggttgcgg ccgcaatcgg gcaaatcgct gaatattcct tttgtctccg
     6001 accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc gctcacggct
     6061 ctggcagtga atgggggtaa atggcactac aggcttgaca gctagctcag tcctaggtat
     6121 aatgctagca aagaggagaa aatgtccaat ttactgaccg tacaccaaaa tttgcctgca
     6181 ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat gttcagggat
     6241 cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg gtcgtgggcg
     6301 gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat
     6361 tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca acatttgggc
     6421 cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag caatgctgtt
     6481 tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag
     6541 gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga aaatagcgat
     6601 cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac cctgttacgt
     6661 atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg tgggagaatg
     6721 ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga gaaggcactt
     6781 agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt agctgatgat
     6841 ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc
     6901 agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg attgatttac
     6961 ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag tgcccgtgtc
     7021 ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat gcaagctggt
     7081 ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag tgaaacaggg
     7141 gcaatggtgc gcctgctgga agatggcgat tagttcagaa cgctcggttg ccgccgggcg
     7201 ttttttatgc cttttatgga ttcatgcaag gaaactaccc ataatacaag aaaagcccgt
     7261 cacgggcttc tcagggcgtt ttatggcg
//
