LOCUS       sequence-482810-        7002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses lambda red machinery and i-creI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7002)
  AUTHORS   Gude S, Bertelsen ABB, Nørholm MHH
  TITLE     Iterative in vivo cut'n'paste of chromosomal loci in Escherichia 
            coli K-12 using synthetic DNA
  JOURNAL   bioRxiv 2025.06.19.660532
REFERENCE   2  (bases 1 to 7002)
  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: "bioRxiv 
            2025.06.19.660532"
COMMENT     Sequence Label: pI-CreIAraRed_SC101_Spec
FEATURES             Location/Qualifiers
     source          1..7002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(122..366)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(367..1047)
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     CDS             complement(1044..1829)
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             complement(1835..2251)
                     /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             2260..2268
                     /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)"
     promoter        complement(2295..2579)
                     /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     complement(2353..2372)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             2606..3484
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     terminator      complement(3535..3566)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        3656..3690
                     /label=BBa_J23116
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.16"
     promoter        4264..4367
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4368..5159
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5781..6003
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             6051..7001
                     /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"
ORIGIN
        1 gttcttatgg ctcttgtatc tatcagtgaa gcatcaagac taacaaacaa aagtagaaca
       61 actgttcacc gttacatatc aaagggaaaa ctgtccatac ccatggattc ttcgtctgtt
      121 tctactggta ttggcacaaa cctgattcca atttgagcaa ggctatgtgc catctcgata
      181 ctcgttctta actcaacaga agatgctttg tgcatacagc ccctcgttta ttatttatct
      241 cctcagccag ccgctgtgct ttcagtggat ttcggataac agaaaggccg ggaaataccc
      301 agcctcgctt tgtaacggag tagacgaaag tgattgcgcc tacccggata ttatcgtgag
      361 gatgcgtcat cgccattgct ccccaaatac aaaaccaatt tcagccagtg cctcgtccat
      421 tttttcgatg aactccggca cgatctcgtc aaaactcgcc atgtactttt catcccgctc
      481 aatcacgaca taatgcaggc cttcacgctt catacgcggg tcatagttgg caaagtacca
      541 ggcatttttt cgcgtcaccc acatgctgta ctgcacctgg gccatgtaag ctgactttat
      601 ggcctcgaaa ccaccgagcc ggaacttcat gaaatcccgg gaggtaaacg ggcatttcag
      661 ttcaaggccg ttgccgtcac tgcataaacc atcgggagag caggcggtac gcatactttc
      721 gtcgcgatag atgatcgggg attcagtaac attcacgccg gaagtgaatt caaacagggt
      781 tctggcgtcg ttctcgtact gttttcccca ggccagtgct ttagcgttaa cttccggagc
      841 cacaccggtg caaacctcag caagcagggt gtggaagtag gacattttca tgtcaggcca
      901 cttctttccg gagcggggtt ttgctatcac gttgtgaact tctgaagcgg tgatgacgcc
      961 gagccgtaat ttgtgccacg catcatcccc ctgttcgaca gctctcacat cgatcccggt
     1021 acgctgcagg ataatgtccg gtgtcatgct gccaccttct gctctgcggc tttctgtttc
     1081 aggaatccaa gagcttttac tgcttcggcc tgtgtcagtt ctgacgatgc acgaatgtcg
     1141 cggcgaaata tctgggaaca gagcggcaat aagtcgtcat cccatgtttt atccagggcg
     1201 atcagcagag tgttaatctc ctgcatggtt tcatcgttaa ccggagtgat gtcgcgttcc
     1261 ggctgacgtt ctgcagtgta tgcagtattt tcgacaatgc gctcggcttc atccttgtca
     1321 tagataccag caaatccgaa ggccagacgg gcacactgaa tcatggcttt atgacgtaac
     1381 atccgtttgg gatgcgactg ccacggcccc gtgatttctc tgccttcgcg agttttgaat
     1441 ggttcgcggc ggcattcatc catccattcg gtaacgcaga tcggatgatt acggtccttg
     1501 cggtaaatcc ggcatgtaca ggattcattg tcctgctcaa agtccatgcc atcaaactgc
     1561 tggttttcat tgatgatgcg ggaccagcca tcaacgccca ccaccggaac gatgccattc
     1621 tgcttatcag gaaaggcgta aatttctttc gtccacggat taaggccgta ctggttggca
     1681 acgatcagta atgcgatgaa ctgcgcatcg ctggcatcac ctttaaatgc cgtctggcga
     1741 agagtggtga tcagttcctg tgggtcgaca gaatccatgc cgacacgttc agccagcttc
     1801 ccagccagcg ttgcgagtgc agtactcatt cgttttatac ctctgaatca atatcaacct
     1861 ggtggtgagc aatggtttca accatgtacc ggatgtgttc tgccatgcgc tcctgaaact
     1921 caacatcgtc atcaaacgca cgggtaatgg attttttgct ggccccgtgg cgttgcaaat
     1981 gatcgatgca tagcgattca aacaggtgct ggggcaggcc tttttccatg tcgtctgcca
     2041 gttctgcctc tttctcttca cgggcgagct gctggtagtg acgcgcccag ctctgagcct
     2101 caagacgatc ctgaatgtaa taagcgttca tggctgaact cctgaaatag ctgtgaaaat
     2161 atcgcccgcg aaatgccggg ctgattagga aaacaggaaa gggggttagt gaatgctttt
     2221 gcttgatctc agtttcagta ttaatatcca ttttttataa cctccttaga gctcgaattc
     2281 ccaaaaaaac gggtatggag aaacagtaga gagttgcgat aaaaagcgtc aggtaggatc
     2341 cgctaatctt atggataaaa atgctatggc atagcaaagt gtgacgccgt gcaaataatc
     2401 aatgtggact tttctgccgt gattatagac acttttgtta cgcgtttttg tcatggcttt
     2461 ggtcccgctt tgttacagaa tgcttttaat aagcggggtt accggtttgg ttagcgagaa
     2521 gagccagtaa aagacgcagt gacggcaatg tctgatgcaa tatggacaat tggtttcttc
     2581 tctgaatggc gggagtatga aaagtatggc tgaagcgcaa aatgatcccc tgctgccggg
     2641 atactcgttt aatgcccatc tggtggcggg tttaacgccg attgaggcca acggttatct
     2701 cgattttttt atcgaccgac cgctgggaat gaaaggttat attctcaatc tcaccattcg
     2761 cggtcagggg gtggtgaaaa atcagggacg agaatttgtt tgccgaccgg gtgatatttt
     2821 gctgttcccg ccaggagaga ttcatcacta cggtcgtcat ccggaggctc gcgaatggta
     2881 tcaccagtgg gtttactttc gtccgcgcgc ctactggcat gaatggctta actggccgtc
     2941 aatatttgcc aatacggggt tctttcgccc ggatgaagcg caccagccgc atttcagcga
     3001 cctgtttggg caaatcatta acgccgggca aggggaaggg cgctattcgg agctgctggc
     3061 gataaatctg cttgagcaat tgttactgcg gcgcatggaa gcgattaacg agtcgctcca
     3121 tccaccgatg gataatcggg tacgcgaggc ttgtcagtac atcagcgatc acctggcaga
     3181 cagcaatttt gatatcgcca gcgtcgcaca gcatgtttgc ttgtcgccgt cgcgtctgtc
     3241 acatcttttc cgccagcagt tagggattag cgtcttaagc tggcgcgagg accaacgtat
     3301 cagccaggcg aagctgcttt tgagcaccac ccggatgcct atcgccaccg tcggtcgcaa
     3361 tgttggtttt gacgatcaac tctacttctc gcgggtattt aaaaaatgca ccggggccag
     3421 cccgagcgag ttccgtgccg gttgtgaaga aaaagtgaat gatgtagccg tcaagttgtc
     3481 ataataaatc gatgcaggtg gcacttttcg gggaaatgtg ccggatcccc ctcaagtcaa
     3541 aagcctccgg tcggaggttt ttgactttct gctatggagg tcaggtatga tttaaatggt
     3601 cagtattgag cgatatctag agaattcgtc aacgaattca agcttaaggt tgaaattgac
     3661 agctagctca gtcctaggga ctatgctagc aaacactact gctctttaac aatttatcag
     3721 atccataagg aggaacataa tgcactcgag cacaaaatat aataaagagt tcttactcta
     3781 cttagcaggg tttgtagacg gtgacggtag cataatcgct caaattaagc ctaatcagtc
     3841 ttataaattt aagcatcagc tatcactcac gttccaagtc acgcaaaaga cacagaggcg
     3901 ttggttttta gacaaattag tggatgaaat tggggttggt tatgtaagag ataggggtag
     3961 cgtttcggat tatattctaa gcgaaatcaa gcctttgcat aattttttaa cacaactaca
     4021 accttttcta aaactaaaac aaaaacaagc aaatttagtt ttaaaaatta ttgaacaact
     4081 tccgtcagca aaagaatccc cggacaaatt cttagaagtt tgtacatggg tggatcaaat
     4141 tgcagctctg aatgattcga acacgcgtaa aacaacttct gaaaccgttc gtgctgtgct
     4201 agacagttta tccggataag atatcattca ggacgagcct cagactccag cgtaactgga
     4261 ctggcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4321 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agggaagcgg
     4381 tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag cgccatctcg
     4441 aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc ctgaagccac
     4501 acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca acgcggcgag
     4561 ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag attctccgcg
     4621 ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat ccagctaagc
     4681 gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc ttcgagccag
     4741 ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat agcgttgcct
     4801 tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg
     4861 cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa
     4921 atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga
     4981 aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac
     5041 ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc
     5101 agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataac
     5161 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5221 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5281 tttcgttcca ctgagcgtca gaccccgttg atgataccgc tgccttactg ggtgcattag
     5341 ccagtctgaa tgacctgtca cgggataatc cgaagtggtc agactggaaa atcagagggc
     5401 aggaactgct gaacagcaaa aagtcagata gcaccacata gcagacccgc cataaaacgc
     5461 cctgagaagc ccgtgacggg cttttcttgt attatgggta gtttccttgc atgaatccat
     5521 aaaaggcgcc tgtagtgcca tttaccccca ttcactgcca gagccgtgag cgcagcgaac
     5581 tgaatgtcac gaaaaagaca gcgactcagg tgcctgatgg tcggagacaa aaggaatatt
     5641 cagcgatttg cccgattgcg gccgcaaccg agcttgcgag ggtgctactt aagcctttag
     5701 ggttttaagg tctgttttgt agaggagcaa acagcgtttg cgacatcctt ttgtaatact
     5761 gcggaactga ctaaagtagt gagttataca cagggctggg atctattctt tttatctttt
     5821 tttattcttt ctttattcta taaattataa ccacttgaat ataaacaaaa aaaacacaca
     5881 aaggtctagc ggaatttaca gagggtctag cagaatttac aagttttcca gcaaaggtct
     5941 agcagaattt acagataccc acaactcaaa ggaaaaggac tagtaattat cattgactag
     6001 cccatctcaa ttggtatagt gattaaaatc acctagacca attgagatgt atgtctgaat
     6061 tagttgtttt caaagcaaat gaactagcga ttagtcgcta tgacttaacg gagcatgaaa
     6121 ccaagctaat tttatgctgt gtggcactac tcaaccccac gattgaaaac cctacaagga
     6181 aagaacggac ggtatcgttc acttataacc aatacgttca gatgatgaac atcagtaggg
     6241 aaaatgctta tggtgtatta gctaaagcaa ccagagagct gatgacgaga actgtggaaa
     6301 tcaggaatcc tttggttaaa ggctttgaga ttttccagtg gacaaactat gccaagttct
     6361 caagcgaaaa attagaatta gtttttagtg aagagatatt gccttatctt ttccagttaa
     6421 aaaaattcat aaaatataat ctggaacatg ttaagtcttt tgaaaacaaa tactctatga
     6481 ggatttatga gtggttatta aaagaactaa cacaaaagaa aactcacaag gcaaatatag
     6541 agattagcct tgatgaattt aagttcatgt taatgcttga aaataactac catgagttta
     6601 aaaggcttaa ccaatgggtt ttgaaaccaa taagtaaaga tttaaacact tacagcaata
     6661 tgaaattggt ggttgataag cgaggccgcc cgactgatac gttgattttc caagttgaac
     6721 tagatagaca aatggatctc gtaaccgaac ttgagaacaa ccagataaaa atgaatggtg
     6781 acaaaatacc aacaaccatt acatcagatt cctacctaca taacggacta agaaaaacac
     6841 tacacgatgc tttaactgca aaaattcagc tcaccagttt tgaggcaaaa tttttgagtg
     6901 acatgcaaag taagtatgat ctcaatggtt cgttctcatg gctcacgcaa aaacaacgaa
     6961 ccacactaga gaacatactg gctaaatacg gaaggatctg ag
//
