LOCUS       sequence-429441-        5053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 Bxb1 integrase with mutated BsaI sites, GG1 and GG4 BsaI 
            sites for golden gate assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5053)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 5053)
  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"
COMMENT     Sequence Label: L0I80_Bxb1mut(noBsaI)_GG1-GG4
FEATURES             Location/Qualifiers
     source          1..5053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             17..1519
                     /codon_start=1
                     /transl_table=11
                     /gene="Bxb1"
                     /product="Bxb1 integrase"
                     /label=Bxb1 integrase
                     /note="Bxb1 large serine integrase from Mycobacterium 
                     bacteriophage Bxb1 (Ghosh et al. 2003)."
                     /protein_id="AFF61396.1"
                     /translation="MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLD
                     VSGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLV
                     VSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGY
                     LPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQG
                     REPQGREWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
                     LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
                     AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAG
                     SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
                     NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     primer_bind     complement(1608..1627)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1609..1627)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1634..1651)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1634..1650)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1643..1665)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1788..2090
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1969..1988
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2439..3233
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2439..3233
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2493..2512)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3103..3122
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3433..3442
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3439..3813
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      3951..4539
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4440..4459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4693..4710
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4827..4848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4863..4868,4869..4886,4887..4893)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4901..4917
                     /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)."
     primer_bind     4906..4928
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4925..4941
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4925..4941
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggatcggagg tctcaaatga gagccctggt agtcatccgc ctgtcccgcg tcaccgatgc
       61 tacgacttca ccggagcgtc agctggagtc ttgccagcag ctctgcgccc agcgcggctg
      121 ggacgtcgtc ggggtagcgg aggatctgga cgtctccggg gcggtcgatc cgttcgaccg
      181 gaagcgcaga ccgaacctgg cccggtggct tgcgttcgag gagcaaccgt ttgacgtgat
      241 cgtggcgtac cgggtagatc ggttgacccg atcgatccgg catcttcagc agctggtcca
      301 ctgggccgag gaccacaaga agctggtcgt ctccgcgacc gaagcgcact tcgatacgac
      361 gacgccgttt gcggcggtcg tcatcgcgct tatgggaacg gtggcgcaga tggaattaga
      421 agcgatcaaa gagcggaacc gttcggctgc gcatttcaat atccgcgccg ggaaataccg
      481 agggtccctg ccgccgtggg gatacctgcc tacgcgcgtg gacggggagt ggcgactggt
      541 gccggaccct gtgcagcgag agcgcatcct cgaggtgtat caccgcgtcg tcgacaacca
      601 cgagccgctg catctggtgg cccacgacct gaaccggcgt ggtgtcctgt cgccgaagga
      661 ctacttcgcg cagctgcaag gccgcgagcc gcagggccgg gagtggtcgg ctaccgcgct
      721 gaagcgatcg atgatctccg aggcgatgct cgggtacgcg actctgaacg gtaagaccgt
      781 ccgagacgac gacggagccc cgctggtgcg ggctgagccg atcctgaccc gtgagcagct
      841 ggaggcgctg cgcgccgagc tcgtgaagac ctcccgggcg aagcccgcgg tgtctacccc
      901 gtcgctgctg ctgcgggtgt tgttctgcgc ggtgtgcggg gagcccgcgt acaagttcgc
      961 cgggggagga cgtaagcacc cgcgctaccg ctgccgctcg atggggttcc cgaagcactg
     1021 cgggaacggc acggtggcga tggccgagtg ggacgcgttc tgcgaggagc aggtactgga
     1081 tctgctcggg gacgcggagc gtctggagaa agtctgggta gcgggctcgg actccgcggt
     1141 cgaactcgcg gaggtgaacg cggagctggt ggacctgacg tcgctgatcg gctccccggc
     1201 ctaccgggcg ggctctccgc agcgagaagc actggatgcc cgtattgcgg cgctggccgc
     1261 gcggcaagag gagctggagg gcctggaggc tcgcccgtct ggctgggagt ggcgcgaaac
     1321 cgggcagcgg ttcggggact ggtggcggga gcaggacacc gcggcaaaga acacctggct
     1381 tcggtcgatg aacgttcggc tgacgttcga cgtccgcggc gggctgactc gcacgatcga
     1441 cttcggggat cttcaggagt acgagcagca tctcaggctc ggcagcgtgg tcgaacggct
     1501 acacaccggg atgtcgtaat aagctttgag acctcatcac gcctgaattc tgcagatatc
     1561 catcacactg gcggccgctc gagcatgcat ctagagggcc caattcgccc tatagtgagt
     1621 cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     1681 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     1741 cccgcaccga tcgcccttcc caacagttgc gcagcctata cgtacggcag tttaaggttt
     1801 acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg
     1861 acacgccggg gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag
     1921 tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca
     1981 ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat ctcagccacc
     2041 gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggca
     2101 tgagattatc aaaaaggatc ttcacctaga tccttttcac gtagaaagcc agtccgcaga
     2161 aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg gaaaacgcaa
     2221 gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta gactgggcgg
     2281 ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt aaggttggga
     2341 agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat
     2401 caagctctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2461 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2521 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2581 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     2641 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2701 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2761 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     2821 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     2881 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     2941 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     3001 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3061 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3121 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3181 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgaattatta
     3241 acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3301 gcatcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3361 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatag
     3421 cacgtgagga gggccaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
     3481 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
     3541 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
     3601 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
     3661 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
     3721 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
     3781 tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc taaaacttca tttttaattt
     3841 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3901 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3961 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4021 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4081 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4141 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4201 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4261 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4321 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4381 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4441 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4501 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4561 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4621 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4681 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     4741 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4801 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     4861 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     4921 cacacaggaa acagctatga ccatgattac gccaagctat ttaggtgacg cgttagaata
     4981 ctcaagctat gcatcaagct tggtaccgag ctcggatcca ctagtaacgg ccgccagtgt
     5041 gctggaattc agg
//
