LOCUS       sequence-436186-        4667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PrACT 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 4667)
  TITLE     Regulating transcription factors in Yarrowia lipolytica
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4667)
  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            "Unpublished"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: pCfb12622
FEATURES             Location/Qualifiers
     source          1..4667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      14..23
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             20..394
                     /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      532..1120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1021..1040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1274..1291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1408..1429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1444..1449,1450..1467,1468..1474)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1482..1498
                     /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     1487..1509
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1506..1522
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1506..1522
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1540..1558
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1540..1557
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     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 gcacgtgagg agggccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
       61 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
      121 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
      181 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
      241 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
      301 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
      361 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc atttttaatt
      421 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      661 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1141 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     1201 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     1261 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     1321 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     1381 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     1441 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     1501 tcacacagga aacagctatg accatgatta cgccaagcta tttaggtgac actatagaat
     1561 actcaagcta tgcatcaagc ttggtaccga gctcggatcc actagtaacg gccgccagtg
     1621 tgctggaatt cgcccttgct cttctcattt ttaatttgtg tggttgttgg tagcaaagag
     1681 atttgtcgat gaaataccga tggaggagag gagaaaagta ggagttggca aacacaaaat
     1741 tggggtgcgc tggctcgtct gggttagggt tagttatgtc gcactgtcca gcacaccaca
     1801 cagcactcac ggttgggaag taacggagac aaggccacat ctcaaaggta aaaaagcact
     1861 gccgaggaat aggggaaggt ggctggtgga gtagggttag ttatgtcgca ctgtccagca
     1921 caccacacag cactcacggt tgggaagtaa cggagacaag gccacatctc aaaggtaaaa
     1981 aagcactgcc gaggaatagg ggaaggtggc tggtggaggg ggtattagga ctggtaaatg
     2041 agccgcttaa ccactggtat ttggtattct agaggaattg aaatcgatcc gaaagttaca
     2101 gagctctagc ccgtctggat tgccctctaa cagcaccgtc ttctcctgtc tttgccaagc
     2161 acactccctt ccaatatact gttatgtacg aaactgtgtg tgtgtgtgtg tgtgtgtgtg
     2221 tgtattggca tatacgattt gactgacaaa aaaaagccta cctctatctc ccccaagctt
     2281 tttggggtcc tctcggtgct aaccactgta ccggagatta tgtcaatgtt gcggtatatt
     2341 ttgaaagtgg tgggttggtg tgcggtgagg gagagagtgg gaggggtgaa aggcgtggat
     2401 ggggtaatta tagcctcggt aatatctccg atatccgact ctcatctgcc ctcctctaat
     2461 ggcacctcct gaataatatc ggaccccggt aggcgtattt ttgcttactc gctgcttggt
     2521 gcgaatctaa aaataccgca ctgtggttga cccctaattt ttagtgcaga gccatatatg
     2581 cgacctagtg cggcttagca tccatgtgag caggcggtgc aggagagatt gacagcgggg
     2641 agctatttgt gggacagtga gtggctccga ctacaagtat tctttgtgcc tcttttgcca
     2701 gcacaagtaa atgccagcac attgtggaat attgccggtt cgcttccccc ccatctacca
     2761 aacaaggttg gtcaagtctg 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 ttcacaccgc atcaggtggc
     4561 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4621 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataata
//
