LOCUS       sequence-460320-        4473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PrGPD 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 4473)
  TITLE     Regulating transcription factors in Yarrowia lipolytica
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4473)
  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            "Unpublished"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: pCfb12562
FEATURES             Location/Qualifiers
     source          1..4473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      302..890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     791..810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1044..1061
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1178..1199
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1214..1219,1220..1237,1238..1244)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1252..1268
                     /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     1257..1279
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1276..1292
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1276..1292
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1310..1328
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1310..1327
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2432..2451)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2433..2451)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2458..2475)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2458..2474)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2467..2489)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2612..2914
                     /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     2793..2812
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3263..4057
                     /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             3263..4057
                     /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(3317..3336)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3927..3946
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4257..4266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4263..4473,1..164)
                     /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"
ORIGIN
        1 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
       61 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
      121 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc
      181 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
      241 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
      301 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
      361 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      421 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
      481 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      541 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      601 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      661 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      721 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      781 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      841 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      901 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
      961 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     1021 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     1081 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     1141 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     1201 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     1261 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcta tttaggtgac
     1321 actatagaat actcaagcta tgcatcaagc ttggtaccga gctcggatcc actagtaacg
     1381 gccgccagtg tgctggaatt cgcccttgct cttctcattg ttgatgtgtg tttaattcaa
     1441 gaatgaatat agagaagaga agaagaaaaa agattcaatt gagccggcga tgcagaccct
     1501 tatataaatg ttgccttgga cagacggagc aagcccgccc aaacctacgt tcggtataat
     1561 atgttaagct ttttaacaca aaggtttggc ttggggtaac ctgatgtggt gcaaaagacc
     1621 gggcgttggc gagccattgc gcgggcgaat ggggccgtga ctcgcctcaa attcgagggc
     1681 gtgcctcaat tcgtgccccc gtggcttttt cccgccgttt ccgccccgtt tgcaccactg
     1741 cagccgcttc tttggttcgg acaccttgct gcgagctagg tgccttgtgc tacttaaaaa
     1801 gtggcctccc aacaccaaca tgacatgagt gcgtgggcca agacacgttg gcggggtcgc
     1861 agtcggctca atggcccgga aaaaacgctg ctggagctgg ttcggacgca gtccgccgcg
     1921 gcgtatcgat atccgcaagg ttccatggcg ccattgccct ccgtcggcgt ctatcccgca
     1981 acctctaaat agagcgggaa tataacccaa gcttcttttt ttttccttta acacgcacac
     2041 ccccaactat catgttgctg ctgctgtttg actctactct gtggaggggt gctcccaccc
     2101 aacccaacct acaggtggat ccggcgctgt gattggctga taagtctcct atccggacta
     2161 attctgacca atgggacatg cgcgcaggac ccaaatgccg caattacgta accccaacga
     2221 aatgcctacc cctctttgga gcccagcggc cccaaatccc cccaagcagc ccggttctac
     2281 cggcttccat ctccaagcac ccctttctcc acaccccaca aaaagacccg tgcaggacat
     2341 cctactgcgt cgtctgaaga gcaagggcga attctgcaga tatccatcac actggcggcc
     2401 gctcgagcat gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact
     2461 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     2521 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     2581 ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga
     2641 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg
     2701 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
     2761 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
     2821 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
     2881 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag
     2941 gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg
     3001 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca
     3061 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag
     3121 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa
     3181 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga
     3241 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3301 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3361 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3421 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     3481 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3541 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3601 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3661 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3721 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3781 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3841 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3901 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3961 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4021 catcgccttc tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct
     4081 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt
     4141 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4201 atccgctcat gagacaataa ccctgataaa tgcttcaata atagcacgtg aggagggcca
     4261 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
     4321 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
     4381 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
     4441 acaacaccct ggcctgggtg tgggtgcgcg gcc
//
