LOCUS       sequence-353782-        6720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector to create landing pad 2 (LP2) by homologous recombination in 
            Aspergillus nidulans IS1 site. The strain A. nidulans LP2 is used 
            for recombinase mediated chromosomal integration..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6720)
  AUTHORS   Roux I, Chooi YH
  TITLE     Cre/lox-Mediated Chromosomal Integration of Biosynthetic Gene 
            Clusters for Heterologous Expression in Aspergillus nidulans.
  JOURNAL   ACS Synth Biol. 2022 Feb 16. doi: 10.1021/acssynbio.1c00458.
  PUBMED    35168324
REFERENCE   2  (bases 1 to 6720)
  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: "ACS Synth 
            Biol. 2022 Feb 16. doi: 10.1021/acssynbio.1c00458."
COMMENT     Sequence Label: pGem-LP2-loxP-bar-lox2272-71
FEATURES             Location/Qualifiers
     source          1..6720
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        1323..1682
                     /label=trpC promoter
                     /note="promoter for Aspergillus nidulans trpC"
     CDS             1705..2256
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     protein_bind    complement(2648..2681)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(3845..3863)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3846..3863)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3881..3897)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3881..3897)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3894..3916)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3905..3921
                     /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(3929..3935,3936..3953,3954..3959))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3974..3995
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4112..4129)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4283..4871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4363..4382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5042..5833,5834..5902))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5665..5684
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5903..6007)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6085..6540)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6222..6243)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6434..6453
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6666..6688
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6680..6697
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6681..6697
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6704..6720,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6704..6720,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg gacatctctc
       61 atggctcgcc ctcgtgtcaa accttctcgc ctgctcacac aatcccaggg cccaatgatg
      121 atgccaactc agcagctata ttctcatgct gggacctcat tcgtaaagca aaaccgagag
      181 ttcataccat ggaagaaacg agcggtctgt tcgatcggca tcaacaagtg ttcctccggg
      241 ttatctgtga tttcatagag acagggtact ccgtccgatg ggcccttctt aattgtatgt
      301 ggtatggggt gccacagtct cgaaagagat tgattatcat tgcgtcaggg tatgcgtgct
      361 caagattcga tgctgactca tgactaacat cggtcctttt taggcctggg gaaagcctcc
      421 cacggctgcc aaggcctact catggcctgc cagggtccgg gctgcgtgat cttacaacaa
      481 tcagccaggc aattcgcgat atccccactg gaagtcccga tcacgacgtc gttgccgcac
      541 ggggtcgcgg agttcataat cgaagagctc cattcgacgg aaaccgacaa gctcgcacaa
      601 taacatgcgg cggtggggac aattatcatc cctctggatt gcgcggtttt accctcaggg
      661 agtttgcttg tctgcaaacc tttcctctgg gctttcggtt cctgggcggt aggacgcagg
      721 taaagaggca gatcggcaat gcagtgcctc ccctactggc caaagctgtc tttaaggaaa
      781 tcatcaggtc gctgcaagac accgatgagc gtgaattgca cgaaaaccaa tgatcgccat
      841 tagggtcctg aggaaccaca tgctctggac ctagccgcgg gattgcagaa cgccgtccaa
      901 ccgtggagac ctcggcgagc gatcggaagc gataacacgc tggacaacgg gaagaggctc
      961 agtctgttct cagccaagac agatagtagt ggcagatctt ccaattgaac gtttgatgat
     1021 tgctggtctg tgtaaagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     1081 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtacagc
     1141 tagctaccgt tcgtatagga tactttatac gaagttatga tctataatgt gagtatacgt
     1201 gattaagcac acaaaggcag cttggagtat gtctgttatt aatttcacag gtagttctgg
     1261 tccattggtg aaagtttgcg gcttgcagag cacagaggcc gcagaatgtg ctctagactc
     1321 gacagaagat gatattgaag gagcactttt tgggcttggc tggagctagt ggaggtcaac
     1381 aatgaatgcc tattttggtt tagtcgtcca ggcggtgagc acaaaatttg tgtcgtttga
     1441 caagatggtt catttaggca actggtcaga tcagccccac ttgtagcagt agcggcggcg
     1501 ctcgaagtgt gactcttatt agcagacagg aacgaggaca ttattatcat ctgctgcttg
     1561 gtgcacgata acttggtgcg tttgtcaagc aaggtaagtg aacgacccgg tcataccttc
     1621 ttaagttcgc ccttcctccc tttatttcag attcaatctg acttacctat tctacccaag
     1681 caaagcttcg attaggaagt aaccatgagc ccagaacgac gcccggccga catccgccgt
     1741 gccaccgagg cggacatgcc ggcggtctgc accatcgtca accactacat cgagacaagc
     1801 acggtcaact tccgtaccga gccgcaggaa ccgcaggagt ggacggacga cctcgtccgt
     1861 ctgcgggagc gctatccctg gctcgtcgcc gaggtggacg gcgaggtcgc cggcatcgcc
     1921 tacgcgggcc cctggaaggc acgcaacgcc tacgactgga cggccgagtc gaccgtgtac
     1981 gtctcccccc gccaccagcg gacgggactg ggctccacgc tctacaccca cctgctgaag
     2041 tccctggagg cacagggctt caagagcgtg gtcgctgtca tcgggctgcc caacgacccg
     2101 agcgtgcgca tgcacgaggc gctcggatat gccccccgcg gcatgctgcg ggcggccggc
     2161 ttcaagcacg ggaactggca tgacgtgggt ttctggcagc tggacttcag cctgccggta
     2221 ccgccccgtc cggtcctgcc cgtcaccgag atctgatccg tcgacctgca gatcgttcaa
     2281 acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca
     2341 tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc atagtaccga
     2401 gaaactagtg gatctaaaca taaaatctgt aaaataacaa gatgtaaaga taatgctaaa
     2461 tcatttggct ttttgattga ttgtacagga aaatatacat cgcagggggt tgacttttac
     2521 catttcaccg caatggaatc aaacttgttg aagagaatgt tcacaggcgc atacgctaca
     2581 atgacccgat tcttgctagc cttttctcgg tcttgcaaac aaccgccgcg gccggtcgag
     2641 ggacctaata acttcgtata gcatacatta tacgaagtta tattaagggt tccggatcgc
     2701 ggcggttgat tgagtcgaga tagtcgagat ggccgggtca taggaacgct ggagtggtag
     2761 atgcggggta cggtgggaag cgccatctgg ggcacccgtt gaccagcttg gaagggccga
     2821 aaagcgctac aatcactaaa gagcacatta aggcgagtcc gattcaaagg acgtaaagca
     2881 gcttgagcaa gatgccaaat tccgagatcg gatggtaaaa tcaccgagtg taaccggcga
     2941 gcagctatgt actccttacg gtgtgcgggt aaatgcccgg cccagaggcg taaaagccac
     3001 ggcctcggct cagtcggcca accgtcaatc cctccctccc agagactcag ccgacccttc
     3061 ttcttctctc cctctccctt ccttccgacc aggcgatttc tttcttctcc ttctgacatc
     3121 aattgggtat tgcaatctca atcctatctc tgtgtccttt ctcttctctt cactatgtct
     3181 gcctcttcag cgctgagacg gctgacccac aggtctcccc gcaatctcca gtcccgcatc
     3241 gcttcgctct cccgcacctc ttgttcccct tcgtcgacgt tcaactccta cagagcttct
     3301 tcatctcctg ctttacctcg tgcttcgaga ttctcaacaa tggcttctct acgatcggcg
     3361 gcgccaattc ccgcgtcctc ccaagtggac tatgatcctg aaatcaagga catggccgac
     3421 tacatccaca actacaagat cgactctgat ctcgcggtat gagctccaat tgagaaccca
     3481 attatggatt gatggggtaa tacgcaattg gtactgatta aatacagttc gacaccgccc
     3541 gcctggttct ccttgatact cttggatgtg gtctggaagc tctgaagttc aaggagtgca
     3601 ccaagctcct tggaccggtt gttgatggca ctgtcgttcc taacggcact cgcgttcctg
     3661 gtactcccta ccagctagac cctgtaaatg gtgccttcaa catcggtgca atgatccgct
     3721 ggctcgatta caatgactgc tggctcgctg ctgagtgggg tcatccttcc gacaacttgg
     3781 gcggccgcct gcaggtcgac catatgggag agctcccaac gcgttggatg catagcttga
     3841 gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3901 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3961 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4021 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4081 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4141 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4201 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4261 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4321 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4381 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4441 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4501 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4561 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4621 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4681 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4741 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4801 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4861 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4921 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4981 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5041 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5101 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5161 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5221 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5281 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5341 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5401 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5461 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5521 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5581 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5641 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5701 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5761 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5821 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5881 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5941 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6001 ttccgcgcac atttccccga aaagtgccac ctgatgcggt gtgaaatacc gcacagatgc
     6061 gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt
     6121 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6181 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6241 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6301 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6361 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     6421 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     6481 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     6541 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     6601 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     6661 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata
//
