LOCUS       sequence-435740-        5051 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 5051)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 5051)
  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..5051
                     /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
                     REPQGREWSATALKRSMISGAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
                     LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
                     AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAG
                     SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
                     NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     primer_bind     complement(1606..1625)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1607..1625)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1632..1649)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1632..1648)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1641..1663)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1786..2088
                     /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     1967..1986
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2437..3231
                     /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             2437..3231
                     /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(2491..2510)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3101..3120
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3431..3440
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3437..3811
                     /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      3949..4537
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4438..4457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4691..4708
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4825..4846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4861..4866,4867..4884,4885..4891)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4899..4915
                     /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     4904..4926
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4923..4939
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4923..4939
                     /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 agcgttcgag 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 aggatccctg ccgccgtggg gatacctgcc tacgcgcgtg gacggggagt ggcggctggt
      541 gccggaccct gtgcagcgag agcgcatcct cgaggtgtat caccgcgtcg tcgacaacca
      601 cgagccgctg catctggtgg cccacgacct gaaccggcgt ggtgtcctgt cgccgaagga
      661 ctacttcgcg cagctgcaag gccgcgagcc gcagggccgg gagtggtcgg ctaccgcgct
      721 gaagcgatcg atgatctccg gggcgatgct 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 ctgaattctg cagatatcca
     1561 tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg
     1621 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     1681 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1741 cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac
     1801 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     1861 acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     1921 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     1981 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
     2041 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg
     2101 agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa
     2161 cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc
     2221 gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt
     2281 ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     2341 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     2401 agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     2461 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     2521 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     2581 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     2641 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2701 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2761 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2821 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2881 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     2941 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     3001 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3061 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3121 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3181 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac
     3241 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3301 atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3361 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca
     3421 cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
     3481 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
     3541 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
     3601 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
     3661 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
     3721 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
     3781 cgtgcacttc gtggccgagg agcaggactg acacgtgcta aaacttcatt tttaatttaa
     3841 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3901 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3961 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4021 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4081 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4141 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4201 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4261 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4321 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4381 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4441 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4501 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4561 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4621 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4681 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     4741 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4801 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     4861 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     4921 cacaggaaac agctatgacc atgattacgc caagctattt aggtgacgcg ttagaatact
     4981 caagctatgc atcaagcttg gtaccgagct cggatccact agtaacggcc gccagtgtgc
     5041 tggaattcag g
//
