LOCUS       sequence-460324-        4540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PrCYC with the overhangs for SapI restriction site can be insert 
            into the base plasmid of TUNEYALI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4540)
  TITLE     Regulating transcription factors in Yarrowia lipolytica
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4540)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCfb12567
FEATURES             Location/Qualifiers
     source          1..4540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      141..150
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             147..521
                     /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      659..1247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1148..1167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1401..1418
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1535..1556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1571..1576,1577..1594,1595..1601)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1609..1625
                     /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     1614..1636
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1633..1649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1633..1649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1667..1685
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1667..1684
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     RBS             2707..2715
                     /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)"
     primer_bind     complement(2856..2875)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2857..2875)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2882..2899)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2882..2898)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2891..2913)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3036..3338
                     /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     3217..3236
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3687..4481
                     /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             3687..4481
                     /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(3741..3760)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4351..4370
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
       61 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
      121 aataatagca cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt ccggtgctca
      181 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
      241 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
      301 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
      361 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
      421 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
      481 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta aaacttcatt
      541 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
      601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
      661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
      721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
      781 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
      841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
      901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
      961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1141 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1201 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1261 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     1321 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     1381 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     1441 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     1501 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     1561 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     1621 aacaatttca cacaggaaac agctatgacc atgattacgc caagctattt aggtgacact
     1681 atagaatact caagctatgc atcaagcttg gtaccgagct cggatccact agtaacggcc
     1741 gccagtgtgc tggaattcgc ccttgctctt cagacagtag acagattctg gaggacagac
     1801 gcaacacttg gctccaatcg ctatcgctgc tgctgccagc tgtcgcccaa aaaatgtaac
     1861 ccttttagtc atgacggtaa aaagctttcg cgcgtcacta gattaaccag actcaaggtc
     1921 ggtcaatatc ggggtcaatc cacgactgaa caaaacgcac agcagtggaa aaagattgat
     1981 ctgtgctcag agacacattg acagaacggt aagatgcttt cgctggatca gtcaacttaa
     2041 ggagagcacg tgacacagaa aacatgtgtc cggatagagt gtgggtcacg cagaaatcac
     2101 cctcgccgca ctttaccatg acacaaatcc actgtgatgt agagaagagg agatgaatta
     2161 gtcgaaacgt ttttttctct cccaactgaa aaatccagga ttagggtatg tccctatccc
     2221 gtaatatctg cgccacattc aacgccacat ttaagggtta gggttgggtt aggacaaaaa
     2281 aataaaaata aaaaaatatc gtcatcggac ttacattttc gtacgagagt cgtcggcgcc
     2341 gggtcaagac agaacattgg actaatagag ttagggttag ggtgttcccg gtgtttggcg
     2401 aaagataggt gaggacgttt gggattgggg aattggggag gtgacaaggg attgttggtc
     2461 acgtgatgaa caggcatgat cattttgttc ggccacgtga tggattggtt cattttcgct
     2521 gatcacgtga ccgacgtttt ccgatatttt ccgagttgct attaatgcac attttttggc
     2581 taatcaacct gcgcgtttat cgcaacggcc aataaggttt ttagagaaag tttaaacgga
     2641 ccaatcacgg ccgaggcatt acctttggag gcgctagtgg ttgctgcagc cgctgagtgc
     2701 aacatataag gaggttggtc cgctactccc agcctcttct caccacactc acatttctac
     2761 aacacaaaaa ctacaatgag aagagcaagg gcgaattctg cagatatcca tcacactggc
     2821 ggccgctcga gcatgcatct agagggccca attcgcccta tagtgagtcg tattacaatt
     2881 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2941 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     3001 gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac acctataaaa
     3061 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc
     3121 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     3181 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     3241 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     3301 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg agattatcaa
     3361 aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa cggtgctgac
     3421 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     3481 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     3541 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     3601 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agctctgatc
     3661 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3721 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3781 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3841 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     3901 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3961 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4021 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4081 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4141 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4201 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4261 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4321 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4381 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4441 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt
     4501 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
//
