LOCUS       sequence-481606-        3472 bp    DNA     circular SYN 31-JUL-2025
DEFINITION  Recombinase-mediated integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3472)
  TITLE     ONE-STEP tagging: a versatile method for rapid site-specific 
            integration by simultaneous reagent delivery
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3472)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 31, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: attB-CT-mCherry-linker-attB-AG::pUC-GW-Kan
FEATURES             Location/Qualifiers
     source          1..3472
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     657..676
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(750..854)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     922..940
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(978..1000)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1095..2249)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMLEPSSARHWDEDSAPDDPDDGVRRRRQAGLSRATSGASLVQL
                     VHAAGGVAALGAFVLFHDGVVLVVGGDVQLDVDVVGAGQLHGLLGLVGGLDLSVVVAAV
                     LQLQPLLDLALQGAVLGVHPLGGGLPAHGFLLHYGAVGGEVGAAQLHLVDELAVLQRGV
                     LGHGHHAAVLEVHHALPLEALGEGQLQVVGDVGGVLHVGLGAVHELRGQDVPGEGQGAT
                     LGHLQLGGLGALVGAALALALDLELVAVHGALHVHLEAQELLDDGGVILLALAHHPDDP
                     DDGERRRRQAGLVFIPIRVNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     primer_bind     1100..1119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1313..1335
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1327..1344
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1328..1344
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1377..1414
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             1419..2126
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNSAIIKEFLRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1564..1582)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    2157..2194
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     primer_bind     complement(2245..2261)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2245..2261)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2258..2280)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2269..2285
                     /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)."
     promoter        complement(join(2293..2299,2300..2317,2318..2323))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2338..2359
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2476..2493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2647..3235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2727..2746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3406..3472,1..749))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRFAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
       61 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
      121 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
      181 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
      241 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
      301 gcgaaacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
      361 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
      421 aatacctgga atgctgtttt cccagggatc gcagtggtga gtaaccatgc atcatcagga
      481 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
      541 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
      601 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
      661 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
      721 caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta ttgaagcatt
      781 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
      841 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
      901 atcatgacat taacctataa aaataggcgt atcacgaggc ccttttgtct cgcgcgtttc
      961 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agactgtcac agcttgtctg
     1021 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     1081 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     1141 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc
     1201 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     1261 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     1321 cgacgttgta aaacgacggc cagtgaattg acgcgtattg ggatgaagac aaggccggct
     1381 tgtcgacgac ggcgctctcc gtcgtcagga tcatccggat ggtgagcaag ggcgaggagg
     1441 ataactccgc catcatcaag gagttcctgc gcttcaaggt gcacatggag ggctccgtga
     1501 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     1561 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     1621 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     1681 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     1741 gcgtggtgac cgtgacccag gactcctctc tgcaggacgg cgagttcatc tacaaggtga
     1801 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaaa accatgggct
     1861 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     1921 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     1981 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     2041 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     2101 ccggcggcat ggacgagctg tacaagggag gctccggagg tggctctgga aaggccggct
     2161 tgtcgacgac ggcggactcc gtcgtcagga tcatccgggg ccgagtcttc atcccaatgg
     2221 cgcgccgagc ttggctcgag catggtcata gctgtttcct gtgtgaaatt gttatccgct
     2281 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     2341 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2401 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2461 gcgctgttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2521 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2581 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2641 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2701 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2761 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2821 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2881 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2941 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3001 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3061 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3121 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3181 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3241 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3301 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3361 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttaga aaaactcatc
     3421 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat at
//
